JP2019506438A - SMC combination therapy for the treatment of cancer - Google Patents

Abstract

  The present invention includes methods and compositions for enhancing the efficacy of SMCs in the treatment of cancer. In particular, the invention includes methods and compositions for combination therapy comprising SMC and at least a second agent that stimulates one or more apoptotic or immune pathways. The second agent may be, for example, an immunostimulatory or immunomodulatory compound or an oncolytic virus.

Description

  Apoptosis (or programmed cell death) and death of cells by other cell death pathways are regulated by various cellular mechanisms. Apoptosis inhibitor (IAP) proteins, such as X-linked IAP (XIAP) or cellular IAP proteins 1 and 2 (cIAP 1 and 2) include (but are not limited to) for example, the apoptotic pathway in cancer cells It is a regulator of programmed cell death. Other forms of cell death include, but are not limited to, necrosis, necrosis, pyroptosis, and immunogenic cell death. Furthermore, these IAPs regulate various cell signaling pathways through their ubiquitin E3 ligase activity, which may or may not be associated with cell survival. Another regulator of apoptosis is the Smac polypeptide. Smac is a pro-apoptotic protein released from mitochondria in association with cell death. Smac can bind to IAP and antagonize its function. Smac mimetic compounds (SMCs) are non-endogenous pro-apoptotic compounds capable of performing one or more functions or activities of endogenous Smac.

  The prototype XIAP protein directly inhibits the caspase protein, a key initiator and executor in the apoptotic cascade. Thereby, XIAP can block the completion of the apoptotic program. Cellular IAP proteins 1 and 2 are E3 ubiquitin ligases that regulate apoptotic signaling pathways involving immune cytokines. Due to the double loss of cIAP1 and 2, TNFα, TRAIL, and / or IL-1β can be toxic to, for example, most cancer cells. SMCs can inhibit XIAP, cIAP1, cIAP2, or other IAPs, and / or contribute to other pro-apoptotic mechanisms.

  Treatment of cancer by administration of SMC has been proposed. However, SMC alone may not be sufficient to treat certain cancers. There is a need for methods of treating cancer that improve the efficacy of SMC treatment in one or more types of cancer.

Houghton, P. J. et al., Initial testing (stage 1) of LCL 161, a SMAC mimetic, by the Pediatric Preliminary Testing Program. Pediatr Blood Cancer 58: 636-639 (2012) Biochemical Pharmacology 84: 268-277 (2012) Chen, K. F. et al., Inhibition of Bcl-2 improves the effect of LCL 161, a SMAC mimetic, in hepatocellular carcinoma cells. Sun, H. et al., Design, synthesis, and characterization of a potent, nonpeptide, cellpermeable, bivalent Smac mimetic that concurrently targets both the BIR2 and BIR3 domains in XIAP. J Am Chem Soc 129: 15279-15294 (2007) Bertrand, M. J. et al., CIAP1 and cIAP2 facilitate cancer cell survival by functioning as E3 ligands that promote RIP1 ubiquitination. Mol Cell 30: 689-700 (2008) Enwere, E. K. et al., TWEAK and cIAP1 regulate myoblast fusion through the noncanonical NF-kappaB signaling pathway. Sci Signal 5: ra75 (2013) Stojdl, D. F. et al., VSV strains with defects in their ability to shut down innate immunity are potent systemic anti-cancer agents. Cancer Cell 4 (4), pp. 263-275 (2003) Brun, J. et al., Identification of genetically modified Maraba virus as an oncolytic rhabdovirus. Mol Ther 18, pp. 1440-1449 (2010) Le Boeuf, F. et al. Synergistic interaction between oncolytic viruses augments tumor killing. MoI Ther 18, pp. 888-895 (2011) Lun, X. et al., Efficacy and safety / toxicity study of recombinant vaccinia virus JX-594 in two immunocompetent animal models of glioma. Mol Ther 18, pp. 1927 to 1936 (2010) Chou, T. C. & Talaly, P. A simple generalized equations for the analysis of multiple inhibitions of Michaelis-Menten kinetic systems. J Biol Chem 252, pp. 6438-6442 (1977) realtimeprimers.com https://software.broadinstitute.org/morpheus

  The present invention includes compositions and methods for the treatment of cancer by administration of SMCs and immunostimulatory or immunomodulatory agents. SMCs and agents including, but not limited to, SMC in Table 1 and Agents in Table 2 (Table 2), Table 3 (Table 3), and Table 4 (Table 4) Described in

  One aspect of the present invention relates to SMC derived from Table 1 (Table 1), and each drug is independently an immune checkpoint inhibitor (ICI) or a drug derived from Table 2 (Table 2) or It is a composition containing one or more (for example, 2, 3, 4, 5 or more) agents which are agents derived from Table 3 (Table 3) or STING agonists. In some embodiments, the ICI is an ICI derived from Table 4 (Table 4). The SMC and the drug are provided together in an amount sufficient to treat cancer when administered to a patient in need thereof. In some embodiments, the two, three or four agents are from different categories (ie one agent is ICI and one agent is from Table 2) And one agent is from Table 3 (Table 3) and / or one agent is a STING agonist).

  Another aspect of the present invention is a method for treating a patient diagnosed with cancer, wherein the patient has SMCs derived from Table 1 (Table 1) and respective agents independently immune checkpoints. One or more (e.g., 2, 3, 4, 3) which are inhibitors (ICIs) or agents derived from Table 2 (Table 2) or agents derived from Table 3 (Table 3) or STING agonists Administering five or more agents. In some embodiments, the ICI is an ICI derived from Table 4 (Table 4), and the SMC and the agent are administered. In some embodiments, two, three, or four agents are from different categories (ie, one agent is ICI and one agent is from Table 2 and 1) One agent is from Table 3 (Table 3) and / or one agent is a STING agonist), together in an amount sufficient to treat cancer, or Each other is administered within 28 days.

  In some embodiments, the SMC and the agent are administered within 14 days of each other, within 10 days of each other, within 5 days of each other, within 24 hours of each other, within 6 hours of each other, or simultaneously.

  In certain embodiments, the SMC is a monovalent SMC, such as LCL 161, SM-122, GDC-0152 / RG 7419, GDC-0917 / CUDC-427, or SM-406 / AT-406 / Debio1143. In other embodiments, the SMC is a bivalent SMC, such as AEG40826 / HGS1049, OICR720, TL32711 / Birinapant, SM-1387 / APG-1387, or SM-164.

  In a particular embodiment, one of the agents is a TLR agonist derived from Table 2. In certain embodiments, the agent is lipopolysaccharide, peptidoglycan, or lipopeptide. In another embodiment, the agent is a CpG oligodeoxynucleotide, such as CpG-ODN2216. In yet another embodiment, the agent is imiquimod or poly (I: C).

  In a specific embodiment, one of the agents is a virus derived from Table 3. In certain embodiments, the agent is Vesicular stomatitis virus (VSV), such as VSV-M51R, VSV-MΔ51, VSV-IFNβ, or VSV-IFNβ-NIS. In another embodiment, the agent is an adenovirus, malababe cyclovirus, reovirus, rhabdovirus, or vaccinia virus, or a variant thereof. In some embodiments, the agent is Talimogene laharpa lepbek, a herpes simplex virus variant.

  In certain embodiments, one of the agents is ICI. In certain embodiments, the drug is ipilimumab, tremelimumab, penbrolizumab, nivolumab, pizirizumab, AMP-224, AMP-514, AUNP12, PDR001, BGB-A317, REGN 2810, averumab, BMS-935595, atezolizumab, durvalumab, BMS- 986016, LAG 525, IMP 321, MBG 453, lirilumab, or MGA 271.

  In some embodiments, the compositions or methods of the invention include, but are not limited to, a plurality of immunostimulatory agents or immunomodulators, such as interferons, and / or a plurality of SMCs.

  In some embodiments, the compositions or methods of the invention comprise one or more interferon agents, such as a type 1 interferon agent, a type 2 interferon agent, and / or a type 3 interferon agent.

  In any of the methods of the invention, the cancer may be a cancer that is refractory to treatment with SMC in the absence of an immunostimulatory agent or an immunomodulatory agent. In any of the methods of the invention, the treatment may further comprise the administration of a therapeutic agent comprising an interferon.

  In any of the methods of the invention, the cancer may be adrenal cancer, basal cell cancer, biliary cancer, bladder cancer, osteosarcoma, brain cancer, breast cancer, cervical cancer, choriocarcinoma, colon cancer, colorectal cancer Cancer, connective tissue cancer, digestive system cancer, endometrial cancer, epipharyngeal cancer, esophageal cancer, eye cancer, gallbladder cancer, stomach cancer, head and neck cancer, hepatocellular carcinoma, intraepithelial Neoplasia, kidney cancer, laryngeal cancer, leukemia, liver cancer, liver metastasis, lung cancer, lymphoma, melanoma, myeloma, multiple myeloma, neuroblastoma, mesothelioma, glioma, myelodysplasia Syndrome, multiple myeloma, oral cancer, ovarian cancer, childhood cancer, pancreatic cancer, pancreatic endocrine tumor, penile cancer, plasma cell tumor, pituitary adenoma, thymoma, prostate cancer, renal cell cancer, Respiratory cancer, rhabdomyosarcoma, salivary gland cancer, sarcoma, skin cancer, small intestine cancer, stomach cancer, testicular cancer, thyroid cancer, ureteral cancer, and A cancer selected from cancer of the urinal system may.

  The invention further includes compositions comprising SMCs derived from Table 1 (Table 1) and one or more (eg, 2, 3, 4 or more) agents as described above. One of the drugs is a killed virus (killed) such that the SMC and the drug, taken together, are provided to a patient in need thereof in an amount sufficient to treat the cancer. virus), inactivated virus, or a viral vaccine. In certain embodiments, the agent is NRRP or a rabies vaccine. In another embodiment, the present invention provides that the SMC and the drug are provided together in an amount sufficient to treat cancer when administered to a patient in need thereof. A composition comprising SMC derived from Table 1, a first agent that primes an immune response, and a second agent that enhances an immune response. In certain embodiments, one or both of the first and second agents are oncolytic viral vaccines. In another particular embodiment, the first agent is an adenovirus carrying a tumor antigen and the second agent is Maraba-MG1 carrying the same tumor antigen as the adenovirus or Maraba- not carrying a tumor antigen It is a Vecyclovirus such as MG1.

  By "nearby" cells is meant cells that are close enough to the reference cell to receive an immune, inflammatory or pro-apoptotic signal directly or indirectly from the reference cell.

  "Promoting apoptosis or cell death" means increasing the possibility that one or more cells will apoptotic or die. The treatment may be by increasing the likelihood that one or more treated cells become apoptotic, and / or one or more cells in the vicinity of the treated cells may apoptotic or die. By increasing, cell death can be promoted.

  By "endogenous Smac activity" is meant one or more biological functions of Smac that result in the promotion of apoptosis, including at least the inhibition of cIAP1 and cIAP2. Biological functions do not have to occur or be possible in all cells under all conditions, Smac performs biological functions in some cells under certain natural in vivo conditions I wish I could.

  "Smac mimetic compound" or "SMC" refers to one or more components capable of inhibiting cIAP1 and / or cIAP2, for example, small molecule, compound, polypeptide, protein or any complex thereof By composition is meant. Smac mimetic compounds include the compounds listed in Table 1 (Table 1).

  “Inducing an apoptosis program” means increasing the amount, availability, or activity of one or more proteins capable of participating in an IAP-mediated apoptosis pathway, or in an IAP-mediated apoptosis pathway It is meant to cause a change in the protein or protein profile of one or more cells such that one or more proteins that can be involved are primed for involvement in the activity of such pathways. The induction of the apoptotic program does not require the initiation of cell death itself: The induction of the program of apoptosis in a manner which does not result in cell death may synergize with treatment with SMC promoting apoptosis, which results in cell death.

  An "agent" is capable of cumulatively inducing an apoptosis or inflammatory program in one or more cells of a subject, and downstream of this program inhibited by at least cIAP1 and cIAP2 cumulatively. It means the composition of the ingredients. The agent is, for example, a TLR agonist (eg, a compound listed in Table 2 (Table 2)), a virus such as an oncolytic virus (eg, a virus listed in Table 3 (Table 3)), or an immune checkpoint It may be an inhibitor (eg, those listed in Table 4).

  "Treatment of cancer" refers to inducing the death of one or more cancer cells in a subject, or inducing tumor regression and inducing an immune response that can block tumor spread (metastasis) It means to do. Treatment of cancer reduces the severity of one or more symptoms of cancer in a subject, which completely or partially abolishes some or all of the signs and symptoms of cancer in the subject, in the subject The progression of one or more symptoms of cancer may be reduced, or the progression or severity of one or more subsequent symptoms occurring may be mediated.

  A "prodrug" is an inactive form that can be converted to an active form in the body of a subject, eg, in a cell of the subject, by the action of one or more enzymes, chemicals or conditions present in the subject. By therapeutic agent is meant.

  "Low dose" or "low concentration" refers to the lowest standard recommended dose or the lowest standard recommended concentration of a particular compound formulated for a given route of administration for the treatment of any human disease or condition By at least 5% less than (eg, at least 10%, 20%, 50%, 80%, 90%, or even 95%) is meant.

  A "high dose" is at least 5% higher (e.g., at least 10%, 20%, 50%, 100%, 200%, or more) than the highest standard recommended dose of the particular compound for treatment of any human disease or condition. %, Or even 300%).

  An "immune checkpoint inhibitor" prevents immune cells from being switched off by cancer cells by either antagonistically blocking the corresponding receptor or by binding to its ligand, thus allowing the tumor to It means a cancer treatment drug that reestablishes the ability of the immune system to attack.

FIG. 6 is a set of graphs and images showing that SMC synergizes with oncolytic rhabdovirus to induce cancer cell death. The entire panel of FIG. 1 is representative of data from at least three independent experiments using biological replicates (n = 3). FIG. 1A is a pair of graphs showing the results of Alamar Blue viability assay of cells treated with LCL 161 and VSV Δ51 of increasing MOI. Error bars, mean ± s. D. FIG. 6 is a set of graphs and images showing that SMC synergizes with oncolytic rhabdovirus to induce cancer cell death. The entire panel of FIG. 1 is representative of data from at least three independent experiments using biological replicates (n = 3). FIG. 1B is a set of micrographs of cells treated with LCL 161 and VSV Δ51-GFP at 0.1 MOI. FIG. 6 is a set of graphs and images showing that SMC synergizes with oncolytic rhabdovirus to induce cancer cell death. The entire panel of FIG. 1 is representative of data from at least three independent experiments using biological replicates (n = 3). FIG. 1C is a pair of graphs showing the viability (alamar blue) of cells infected with VSV Δ51 (0.1 MOI) in the presence of increasing concentrations of LCL 161. Error bars, mean ± s. D. FIG. 6 is a set of graphs and images showing that SMC synergizes with oncolytic rhabdovirus to induce cancer cell death. The entire panel of FIG. 1 is representative of data from at least three independent experiments using biological replicates (n = 3). FIG. 1D is a pair of graphs showing data from cells infected with VSV Δ51 for 24 hours. The cell culture supernatant was exposed to UV light to inactivate the virus, then media was applied to fresh cells for viability assay (alamar blue) in the presence of LCL161. Error bars, mean ± s. D. FIG. 6 is a set of graphs and images showing that SMC synergizes with oncolytic rhabdovirus to induce cancer cell death. The entire panel of FIG. 1 is representative of data from at least three independent experiments using biological replicates (n = 3). FIG. 1E is a graph showing the viability of cells co-treated with LCL 161 and non-diffusible virus VSV Δ51 ΔG (0.1 MOI). Error bars, mean ± s. D. FIG. 6 is a set of graphs and images showing that SMC synergizes with oncolytic rhabdovirus to induce cancer cell death. The entire panel of FIG. 1 is representative of data from at least three independent experiments using biological replicates (n = 3). FIG. 1F is a graph and a pair of images of cells which were covered with agarose medium containing LCL 161, inoculated with VSV Δ51-GFP at the center of the wells, and measured infectivity by fluorescence, and cytotoxicity assessed by crystal violet staining. (The images were superimposed; the non-superimposed image is in FIG. 11). Error bars, mean ± s. D. FIG. 6 is a set of graphs and images showing that SMC treatment does not alter cancer cell response to oncolytic virus (OV) infection. The entire panel of FIG. 2 is representative of data from at least three independent experiments using biological replicates. FIG. 2A is a pair of graphs showing data from cells pretreated with LCL 161 and infected with the indicated MOI VSV Δ51. Virus titer was assessed by standard plaque assay. FIG. 6 is a set of graphs and images showing that SMC treatment does not alter cancer cell response to oncolytic virus (OV) infection. The entire panel of FIG. 2 is representative of data from at least three independent experiments using biological replicates. FIG. 2B is a set of a pair of graphs and micrographs captured over time from cells treated with LCL 161 and VSV Δ51-GFP. The graph plots GFP signal number over time. Error bars, mean ± s. D. n = 12. FIG. 6 is a set of graphs and images showing that SMC treatment does not alter cancer cell response to oncolytic virus (OV) infection. The entire panel of FIG. 2 is representative of data from at least three independent experiments using biological replicates. FIG. 2C is a pair of graphs showing data from experiments in which cell culture supernatants from cells treated with LCL 161 and VSV Δ51 were processed for IFNβ by ELISA. Error bars, mean ± s. D. n = 3. FIG. 6 is a set of graphs and images showing that SMC treatment does not alter cancer cell response to oncolytic virus (OV) infection. The entire panel of FIG. 2 is representative of data from at least three independent experiments using biological replicates. FIG. 2D is a pair of graphs showing data from experiments in which cells were treated with LCL 161 and VSVΔ51 for 20 hours, processed for RT-qPCR, and measuring interferon stimulated gene (ISG) expression. Error bars, mean ± s. D. n = 3. FIG. 6 is a set of graphs and images showing that SMC treatment does not alter cancer cell response to oncolytic virus (OV) infection. The entire panel of FIG. 2 is representative of data from at least three independent experiments using biological replicates. FIG. 2E is a pair of images showing immunoblots for STAT1 pathway activation performed on IFNβ-stimulated cells after pre-treatment with LCL161. FIG. 6 is a set of graphs showing that SMC treatment of OV infected cancer cells induces proinflammatory cytokine type 1 interferon (type 1 IFN) and nuclear factor kappa B (NF-κb) dependent production. The entire panel of FIG. 3 is representative of data from at least three independent experiments using biological replicates (n = 3). FIG. 3A is a graph showing Alamar Blue viability assay of cells treated with LCL 161 and VSVΔ51 (0.1 MOI) or IFNβ after transfection of a combination of non-target (NT), TNF-R1 and DR5 siRNA. Error bars, mean ± s. D. FIG. 6 is a set of graphs showing that SMC treatment of OV infected cancer cells induces proinflammatory cytokine type 1 interferon (type 1 IFN) and nuclear factor kappa B (NF-κb) dependent production. The entire panel of FIG. 3 is representative of data from at least three independent experiments using biological replicates (n = 3). FIG. 3B is a graph showing the viability of cells treated with LCL 161 and VSVΔ51G after transfection with NT or IFNAR1 siRNA. Error bars, mean ± s. D. FIG. 6 is a set of graphs showing that SMC treatment of OV infected cancer cells induces proinflammatory cytokine type 1 interferon (type 1 IFN) and nuclear factor kappa B (NF-κb) dependent production. The entire panel of FIG. 3 is representative of data from at least three independent experiments using biological replicates (n = 3). FIG. 3C is a graph showing data from experiments in which cells were pretreated with LCL 161 and infected with VSV Δ51 at 0.5 MOI and cytokine gene expression was measured by RT-qPCR. Error bars, mean ± s. D. FIG. 6 is a set of graphs showing that SMC treatment of OV infected cancer cells induces proinflammatory cytokine type 1 interferon (type 1 IFN) and nuclear factor kappa B (NF-κb) dependent production. The entire panel of FIG. 3 is representative of data from at least three independent experiments using biological replicates (n = 3). FIG. 3D is a chart showing data collected from experiments in which cytokine ELISA was performed on cells treated with LCL 161 and 0.1 MOI of VSV Δ51 after transfection with NT or IFNAR1 siRNA. Error bars, mean ± s. D. FIG. 6 is a set of graphs showing that SMC treatment of OV infected cancer cells induces proinflammatory cytokine type 1 interferon (type 1 IFN) and nuclear factor kappa B (NF-κb) dependent production. The entire panel of FIG. 3 is representative of data from at least three independent experiments using biological replicates (n = 3). FIG. 3E is a graph showing the viability of cells co-treated with LCL 161 and cytokines. Error bars, mean ± s. D. FIG. 6 is a set of graphs showing that SMC treatment of OV infected cancer cells induces proinflammatory cytokine type 1 interferon (type 1 IFN) and nuclear factor kappa B (NF-κb) dependent production. The entire panel of FIG. 3 is representative of data from at least three independent experiments using biological replicates (n = 3). FIG. 3F is a graph showing data from experiments in which cells were pretreated with LCL 161 and stimulated with 250 U / mL (about 20 pg / mL) of IFNβ and cytokine mRNA levels were determined by RT-qPCR. Error bars, mean ± s. D. FIG. 6 is a set of graphs showing that SMC treatment of OV infected cancer cells induces proinflammatory cytokine type 1 interferon (type 1 IFN) and nuclear factor kappa B (NF-κb) dependent production. The entire panel of FIG. 3 is representative of data from at least three independent experiments using biological replicates (n = 3). FIG. 3G is a pair of graphs showing the results of cytokine ELISA performed on cells treated with VCL Δ51 of LCL 161 and 0.1 MOI. FIG. 6 is a set of graphs showing that SMC treatment of OV infected cancer cells induces proinflammatory cytokine type 1 interferon (type 1 IFN) and nuclear factor kappa B (NF-κb) dependent production. The entire panel of FIG. 3 is representative of data from at least three independent experiments using biological replicates (n = 3). FIG. 3H is a graph showing the results of cytokine ELISA performed on cells expressing IKKβ-DN and treated with LCL161 and VSVΔ51 or IFNβ. Error bars, mean ± s. D. FIG. 6 is a set of graphs and images showing that the combination of SMC and OV treatment is effective in vivo and is dependent on cytokine signaling. FIG. 4A is a pair of graphs showing data from experiments in which EMT6-Fluc tumors were treated with 50 mg / kg LCL 161 (po) and 5 × 10 ⁸ PFU of VSV Δ51 (iv). Left panel shows tumor growth. Right panel represents Kaplan-Meier curve showing survival of mice. Error bars, mean ± sem. n = 5 / group. Log rank with Form-Sidak multiple comparisons: ^** , p <0.01; ^*** , p <0.001 Representative data from two independent experiments are shown. FIG. 6 is a set of graphs and images showing that the combination of SMC and OV treatment is effective in vivo and is dependent on cytokine signaling. FIG. 4B is a series of representative IVIS images obtained from the experiment of FIG. 4A. FIG. 6 is a set of graphs and images showing that the combination of SMC and OV treatment is effective in vivo and is dependent on cytokine signaling. FIG. 4C is a set of immunofluorescent images of infection and apoptosis in tumors treated for 24 hours using α-VSV or α-c-caspase-3 antibodies. FIG. 6 is a set of graphs and images showing that the combination of SMC and OV treatment is effective in vivo and is dependent on cytokine signaling. FIG. 4D is a set of immunofluorescent images of infection and apoptosis in tumors treated for 24 hours using α-VSV or α-c-caspase-3 antibodies. FIG. 6 is a set of graphs and images showing that the combination of SMC and OV treatment is effective in vivo and is dependent on cytokine signaling. FIG. 4E is an image showing an immunoblot immunoblotted with the indicated antibodies for protein lysates of tumors from correspondingly treated mice. FIG. 6 is a set of graphs and images showing that the combination of SMC and OV treatment is effective in vivo and is dependent on cytokine signaling. FIG. 4F shows an experiment in which mice bearing EMT6-Fluc tumor were injected with neutralizing TNFα or isotype-matched antibody and then treated with 50 mg / kg of LCL 161 (po) and 5 × 10 ⁸ PFU of VSV Δ51 (iv). It is a pair of graphs which show the data derived from. Left panel shows tumor growth. Right panel represents Kaplan-Meier curve showing survival of mice. Error bars, mean ± sem. SMC α-TNF α, n = 5; SMC α-TNF α, n = 5; Vehicle + VSV Δ51, n = 5; α-TNF α, n = 5; SMC + VSV Δ51 α-TNF α, n = 7; SMC + VSV Δ51 α- IgG, n = 7. Log rank with Holm-Sidak multiple comparisons: ^*** , p <0.001. FIG. 6 is a set of graphs and images showing that the combination of SMC and OV treatment is effective in vivo and is dependent on cytokine signaling. FIG. 4G is a set of representative IVIS images obtained from the experiment of FIG. 4F. FIG. 7 is a series of graphs and images showing that small molecule immunostimulatory agents enhance SMC therapy in a mouse cancer model. FIG. 5A is a graph showing the results of Alamar Blue Viability Assay of EMT6 cells co-cultured with splenocytes in the Transwell system and treated splenocytes separated with LCL 161 and the indicated TLR agonists. Error bars, mean ± s. D. Representative data from at least three independent experiments using biological replicates (n = 3) are shown. FIG. 7 is a series of graphs and images showing that small molecule immunostimulatory agents enhance SMC therapy in a mouse cancer model. FIG. 5B is a pair of graphs showing the results of experiments in which established EMT6-Fluc tumors were treated with SMC (50 mg / kg LCL 161, po) and poly (I: C) (15 μg it or 2.5 mg / kg ip) is there. Left panel shows tumor growth. Right panel represents Kaplan-Meier curve showing survival of mice. Vehicle, vehicle + poly (I: C) ip, n = 4; remaining groups, n = 5. Error bars, mean ± sem. Log rank with Holm-Sidak multiple comparisons: ^** , p <0.01; ^*** , p <0.001. FIG. 7 is a series of graphs and images showing that small molecule immunostimulatory agents enhance SMC therapy in a mouse cancer model. FIG. 5C is a series of representative IVIS images obtained from the experiment of FIG. 5B. FIG. 7 is a series of graphs and images showing that small molecule immunostimulatory agents enhance SMC therapy in a mouse cancer model. FIG. 5D is a pair of graphs showing the results of experiments in which EMT6-Fluc tumors were treated with LCL 161 or a combination of 200 μg (it) and / or 2.5 mg / kg (ip) CpG ODN 2216. Left panel shows tumor growth. Right panel represents Kaplan-Meier curve showing survival of mice. Vehicle, n = 5; SMC, n = 5; Vehicle + CpG ip, n = 5; SMC + CpG ip, n = 7; Vehicle + CpG it, n = 5; SMC + CpG it, n = 8; Vehicle + CpG ip + it, n = 5; SMC + CpG ip + it, n = 8. Error bars, mean ± sem. Log rank with Holm-Sidak multiple comparisons: ^* , p <0.05; ^** , p <0.01; ^*** , p <0.001. FIG. 7 is a series of graphs and images showing that small molecule immunostimulatory agents enhance SMC therapy in a mouse cancer model. FIG. 5E is a series of representative IVIS images obtained from the experiment of FIG. 5D. FIG. 16 is a graph showing the responsiveness of a panel of cancer cells and normal cells to combined treatment with SMC and OV. The indicated cancer cell lines (n = 28) and non-cancerous human cells (primary human skeletal muscle (HSkM) and human fibroblasts (GM38)) were treated with LCL 161 and increasing VSV Δ51 for 48 hours. The dose required to obtain 50% viable cells in the presence of SMC vs vehicle was determined using non-linear regression and plotted as log EC 50 shifts towards increasing sensitivity. Representative data from at least two independent experiments using biological replicates (n = 3) are shown. FIG. 16 is a pair of graphs showing that co-treatment of SMC and OV is highly synergistic in cancer cells. The graph shows Alamar Blue viability of cells treated with serial dilutions (PFU: μM LCL161) of a combined mixture of fixed ratios of VSV Δ51 and LCL 161. Combination coefficient (CI) was calculated using Calcusyn. The plots represent algebraic estimates of CI in function of the fraction of affected cells (Fa). Error bars, mean ± s. E. M. Representative data from three independent experiments using biological replicates (n = 3) are shown. FIG. 6 is a pair of graphs showing that monovalent and divalent SMCs synergize with OV to cause cancer cell death. The graph shows the results of Alamar Blue Viability Assay of cells treated with 5 μM monovalent SMC (LCL 161, SM-122) or 0.1 μM bivalent SMC (AEG 40730, OICR 720, SM-164) and different MOI VSV Δ51 Indicates Error bars, mean ± s. D. Representative data from three independent experiments using biological replicates (n = 3) are shown. FIG. 6 is a set of images and graphs showing that SMC-mediated cancer cell death is promoted by oncolytic viruses. FIG. 9A is a series of images showing the results of a virus diffusion assay of cells spread with 0.7% agarose and 500 PFU of the indicated virus spread in the middle of the well in the presence of vehicle or LCL161. Cytotoxicity was assessed by crystal violet staining. Arrows indicate elongation of cell death area from the origin of OV infection. FIG. 6 is a set of images and graphs showing that SMC-mediated cancer cell death is promoted by oncolytic viruses. FIG. 9B is a set of graphs showing Alamar Blue viability of LCL 161 and cells treated with VSV Δ51 or Maraba-MG1 with increasing MOI. Error bars, mean ± s. D. Representative data from two independent experiments using biological replicates (n = 3) are shown. FIG. 6 is a set of graphs and images showing that cIAP1, cIAP2 and XIAP coordinately protect cancer cells from OV induced cell death. FIG. 10A shows Alamar Blue viability of cells treated with LCL 161 and 0.1 MOI VSV Δ51 for 48 hours after transfection with non-target (NT) siRNA or siRNA targeting cIAP1, cIAP2 or XIAP. Error bars, mean ± s. D. Representative data from three independent experiments using biological replicates (n = 3) are shown. FIG. 6 is a set of graphs and images showing that cIAP1, cIAP2 and XIAP coordinately protect cancer cells from OV induced cell death. FIG. 10B is a representative siRNA efficacy immunoblot for the experiment of FIG. 10A. FIG. 1G is a set of images used for the superimposed image shown in FIG. 1G. The cells were covered with agarose medium containing LCL161, the center of the wells was inoculated with VSVΔ51-GFP, the infectivity was measured by fluorescence, and the cytotoxicity was shown by crystal violet (CV) staining. Note: The bars represent the same size. Image-graph set showing that SMC treatment does not affect the distribution or replication of OV in vivo. FIG. 12A is a set of images showing images from experiments in which EMT6 loaded mice were treated with 50 mg / kg LCL 161 (po) and 5 × 10 ⁸ PFU of firefly luciferase tagged VSV Δ 51 (VSV Δ 51-Fluc) by iv injection. It is. Virus distribution and replication were imaged at 24 and 48 hours using IVIS. Contours indicate the area of the tumor. Representative data from two independent experiments are shown. Arrows indicate spleen infected with VSV Δ51-Fluc. Image-graph set showing that SMC treatment does not affect the distribution or replication of OV in vivo. FIG. 12B is a graph showing data from experiments in which tumors and tissues were homogenized 48 hours after infection and virus titration was performed for each group. Error bars, mean ± s. E. M. 12 is an image showing validation of siRNA-mediated knockdown of non-target (NT), TNFR1, DR5 and IFNAR1 by immunoblotting. FIG. 13A is an immunoblot showing knockdown in samples derived from the experiment of FIG. 3A. 12 is an image showing validation of siRNA-mediated knockdown of non-target (NT), TNFR1, DR5 and IFNAR1 by immunoblotting. FIG. 13B is an immunoblot showing knockdown in samples from the experiment of FIG. 3B. BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is an image and a graph showing that SMC synergizes with OV to induce caspase 8 and RIP-1 dependent apoptosis in cancer cells. The full panel of FIG. 14 shows representative data from three independent experiments using biological replicates. FIG. 14A is a pair of images of immunoblots for caspase and PARP activation performed on cells treated with 1 MOI of VSV Δ51 after being pretreated with LCL 161. BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is an image and a graph showing that SMC synergizes with OV to induce caspase 8 and RIP-1 dependent apoptosis in cancer cells. The full panel of FIG. 14 shows representative data from three independent experiments using biological replicates. FIG. 14B is a series of images showing a micrograph of caspase activation obtained with cells co-treated with LCL 161 and VSV Δ51 in the presence of the caspase 3/7 substrate, DEVD-488. BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is an image and a graph showing that SMC synergizes with OV to induce caspase 8 and RIP-1 dependent apoptosis in cancer cells. The full panel of FIG. 14 shows representative data from three independent experiments using biological replicates. FIG. 14C is a graph plotting the percentage of DEVD-488 positive cells derived from the experiment of FIG. 14B (n = 12). Error bars, mean ± s. D. BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is an image and a graph showing that SMC synergizes with OV to induce caspase 8 and RIP-1 dependent apoptosis in cancer cells. The full panel of FIG. 14 shows representative data from three independent experiments using biological replicates. FIG. 14D is a series derived from experiments in which apoptosis was assessed by microscopy of translocated phosphatidylserine (Annexin V-CF 594) and loss of plasma membrane integrity (YOYO-1) in cells treated with LCL 161 and VSV Δ51. Image. BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is an image and a graph showing that SMC synergizes with OV to induce caspase 8 and RIP-1 dependent apoptosis in cancer cells. The full panel of FIG. 14 shows representative data from three independent experiments using biological replicates. FIG. 14E is a graph plotting the percentage of annexin V-CF 594 positive and YOYO-1 negative apoptotic cells derived from the experiment of FIG. 14D (n = 9). Error bars, mean ± s. D. BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is an image and a graph showing that SMC synergizes with OV to induce caspase 8 and RIP-1 dependent apoptosis in cancer cells. The full panel of FIG. 14 shows representative data from three independent experiments using biological replicates. FIG. 14F is a pair showing Alamar blue viability of cells treated with LCL 161 and 0.1 MOI VSV Δ51 (n = 3) after transfection with non-target (NT) siRNA or siRNA targeting caspase 8 or RIP1 Is a graph of Error bars, mean ± s. D. BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is an image and a graph showing that SMC synergizes with OV to induce caspase 8 and RIP-1 dependent apoptosis in cancer cells. The full panel of FIG. 14 shows representative data from three independent experiments using biological replicates. FIG. 14G is an image of an immunoblot showing representative siRNA efficacy for the experiment of FIG. 14F. FIG. 5 is a set of graphs showing that expression of the TNFα transgene derived from OV further promotes SMC-mediated cancer cell death. FIG. 15A is a pair of graphs showing an Alamar Blue Viability Assay of cells co-treated for 24 hours with 5 μM SMC and VSV Δ51-GFP or VSV Δ51-TNFα of increasing MOI. Error bars, mean ± s. D. FIG. 5 is a set of graphs showing that expression of the TNFα transgene derived from OV further promotes SMC-mediated cancer cell death. It is a graph which shows the typical EC50 shift derived from the experiment of FIG. 15A. The dose required to obtain 50% viable cells in the presence of SMC versus vehicle was determined using non-linear regression and plotted as an EC50 shift. Representative data from three independent experiments using biological replicates (n = 3) are shown. FIG. 6 is a set of images showing that oncolytic viral infection results in enhanced TNFα expression upon SMC treatment. EMT6 cells were co-treated with 5 μM SMC and VSV Δ51-GFP at 0.1 MOI for 24 hours and cells were processed by flow cytometry for the presence of intracellular TNFα. Images show representative data from four independent experiments. FIG. 6 is a pair of graphs and images showing that TNFα signaling is required for type I IFN-induced synergy with SMC treatment. The full panel of FIG. 17 shows representative data from at least three independent experiments using biological replicates (n = 3). FIG. 17A is a graph showing the results of Alamar Blue Viability Assay of EMT6 cells treated with LCL161 and VSVΔ51 (0.1 MOI) or IFNβ after transfection with non-target (NT) or TNF-R1 siRNA. Error bars, mean ± s. D. FIG. 6 is a pair of graphs and images showing that TNFα signaling is required for type I IFN-induced synergy with SMC treatment. The full panel of FIG. 17 shows representative data from at least three independent experiments using biological replicates (n = 3). FIG. 17B is a representative siRNA efficacy blot from the experiment of FIG. 17A. FIG. 6 is a pair of graphs and images showing that TNFα signaling is required for type I IFN-induced synergy with SMC treatment. The full panel of FIG. 17 shows representative data from at least three independent experiments using biological replicates (n = 3). FIG. 17C is a graph showing the viability of EMT6 cells treated with 5 μM SMC and VSVΔ51 or IFNβ after pretreatment with TNFα neutralizing antibody. FIG. 5 is a schematic representation of the synergy of OV-induced type I IFN and SMC in bystander cancer cell death. FIG. 18A is a schematic diagram showing that viral infection in refractory cancer cells results in the production of type 1 IFN and then induces the expression of IFN stimulated genes such as TRAIL. Type 1 IFN stimulation also results in NF-κB dependent production of TNFα. IAP antagonism by SMC treatment results in upregulation of TNFα and TRAIL expression as well as apoptosis of neighboring tumor cells. FIG. 5 is a schematic representation of the synergy of OV-induced type I IFN and SMC in bystander cancer cell death. FIG. 18B is a schematic illustration showing that infection of a single tumor cell results in the activation of the innate antiviral type 1 IFN pathway, resulting in the secretion of type 1 IFN into neighboring cells. Neighboring cells also produce the proinflammatory cytokines TNFα and TRAIL. Single infected cells undergo oncolysis and the remaining tumor mass remains intact. On the other hand, neighboring cells undergo bystander cell death due to SMC treatment as a result of SMC-mediated upregulation of TNFα / TRAIL and promotion of apoptosis upon proinflammatory cytokine activation. SMC and treatment are graphs and blots that show minimal transient weight loss leading to downregulation of cIAP1 / 2. FIG. 19A is a graph showing body weight from LCL 161-treated female BALB / c mice (50 mg / kg LCL 161, po) recorded after a single treatment (day 0). n = 5 / group. Error bars, mean ± s. E. M. SMC and treatment are graphs and blots that show minimal transient weight loss leading to downregulation of cIAP1 / 2. FIG. 19B is a blot of samples from experiments in which EMT6 tumor-bearing mice were treated with 50 mg / kg LCL 161 (po). Tumors were harvested at the times indicated for Western blotting using the indicated antibodies. FIG. 16 is a set of graphs showing that SMC treatment induces transient weight loss in a syngeneic mouse model of cancer. FIG. 20A is a graph showing measurements of mouse weight upon simultaneous treatment of SMC and oncolytic VSV in tumor bearing animals derived from the experiment shown in FIG. 4A. Error bars, mean ± s. E. M. FIG. 16 is a set of graphs showing that SMC treatment induces transient weight loss in a syngeneic mouse model of cancer. FIG. 20B is a graph showing measurements of mouse weight upon co-treatment of SMC and poly (I: C) in tumor-bearing animals from the experiment shown in FIG. 5B. Error bars, mean ± s. E. M. FIG. 16 is a set of graphs showing that SMC treatment induces transient weight loss in a syngeneic mouse model of cancer. FIG. 20C is a graph showing measurements of mouse weight upon co-treatment of SMC and CpG in tumor-bearing animals from the experiment shown in FIG. 5D. Error bars, mean ± s. E. M. FIG. 16 is a series of graphs showing that VSV Δ51 induced cell death in HT-29 cells is promoted by SMC treatment in vitro and in vivo. FIG. 21A is a graph showing data from experiments in which cells were infected with VSV Δ51, cell culture supernatant was exposed to UV light for 1 hour, and applied to new cells at the indicated doses in the presence of LCL 161. Viability was confirmed by Alamar Blue. Error bars, mean ± s. D. FIG. 21A shows representative data from three independent experiments using biological replicates (n = 3). FIG. 16 is a series of graphs showing that VSV Δ51 induced cell death in HT-29 cells is promoted by SMC treatment in vitro and in vivo. FIG. 21B is a graph showing Alamar Blue viability of cells co-treated with LCL 161 and non-diffusible virus VSVΔ51ΔG (0.1 MOI). Error bars, mean ± s. D. FIG. 21B shows representative data from three independent experiments using biological replicates (n = 3). FIG. 16 is a series of graphs showing that VSV Δ51 induced cell death in HT-29 cells is promoted by SMC treatment in vitro and in vivo. FIG. 21C is a pair showing data from experiments in which CD-1 nude mice with established HT-29 tumors were treated with 50 mg / kg of LCL 161 (po) and 1 × 10 ⁸ PFU of VSV Δ51 (it) Is a graph of Vehicle, n = 5; VSVΔ51, n = 6; SMC, n = 6; VSVΔ51 + SMC, n = 7. Left panel shows tumor growth compared to day 0 after treatment. The right panel represents Kaplan-Meier curves showing mouse survival. Error bars, mean ± sem. Log rank with Holm-Sidak multiple comparisons: ^*** , p <0.001. FIG. 16 is a series of graphs showing that VSV Δ51 induced cell death in HT-29 cells is promoted by SMC treatment in vitro and in vivo. FIG. 21D is a graph showing measurements of mouse weight upon simultaneous treatment of SMC and OV in tumor-bearing animals. Error bars, mean ± s. E. M. FIG. 6 is a blot showing that type I IFN signaling is required for SMC-OV synergy in vivo. EMT6 tumor-bearing mice were treated with vehicle or 50 mg / kg LCL 161 for 4 hours and then treated with neutralizing IFNAR1 or isotype antibody for 20 hours. Subsequently, animals were treated with PBS or VSV Δ51 for 18 hours. Tumors were processed for Western blotting using the indicated antibodies. FIG. 6 is a pair of graphs showing that oncolytic infection of innate immune cells results in cancer cell death in the presence of SMC. FIG. 23A shows that immune subpopulations are sorted from splenocytes (CD11b + F4 / 80 +: macrophages; CD11b + Gr1 +: neutrophils; CD11b-CD49b +: NK cells; CD11b-CD49b-: T and B cells), 24 hours Is a graph showing data from experiments infected with 1 MOI of VSV Δ51. Cell culture supernatants were applied to SMC-treated EMT6 cells for 24 hours and EMT6 viability was assessed by Alamar Blue. Error bars, mean ± s. D. FIG. 6 is a pair of graphs showing that oncolytic infection of innate immune cells results in cancer cell death in the presence of SMC. FIG. 23B is a chart showing data from experiments in which bone marrow derived macrophages were infected with VSVΔ51, supernatants were applied to EMT6 cells in the presence of 5 μM SMC, and viability was measured by Alamar Blue. Error bars, mean ± s. D. 8 is a series of images of full length immunoblots. The immunoblot of FIG. 24A relates to FIG. 2E. 8 is a series of images of full length immunoblots. The immunoblot of FIG. 24B relates to FIG. 4E. 8 is a series of images of full length immunoblots. The immunoblot of FIG. 24C relates to FIG. 10B. 8 is a series of images of full length immunoblots. The immunoblot of FIG. 24D relates to FIG. 8 is a series of images of full length immunoblots. The immunoblot of FIG. 24E relates to FIG. 14A. 8 is a series of images of full length immunoblots. The immunoblot of FIG. 24F relates to FIG. 14G. 8 is a series of images of full length immunoblots. The immunoblot of FIG. 24G relates to FIG. 8 is a series of images of full length immunoblots. The immunoblot of FIG. 24H relates to FIG. FIG. 6 is a set of graphs showing that non-replicating rhabdovirus derived particles (NRRP) synergize with SMC to cause cancer cell death. FIG. 25A shows EMT6, DBT, and CT-2A cancer cells with SMC LCL 161 (SMC; EMT 6: 5 μM, DBT and CT-2A: 15 μM) and different numbers of NRRPs for 48 hours (EMT 6) or 72 hours (DBT) , CT-2A), a set of graphs showing data from experiments in which cell viability was assessed by Alamar Blue. FIG. 6 is a set of graphs showing that non-replicating rhabdovirus derived particles (NRRP) synergize with SMC to cause cancer cell death. FIG. 25B is a pair of graphs showing data from experiments in which unfractionated mouse splenocytes were incubated for 24 hours with one particle of NRRP or 250 μM CpG ODN 2216 per cell. Subsequently, the supernatant was applied to EMT6 cells in a dose response manner and 5 μM LCL161 was added. EMT6 viability was assessed 48 hours after treatment with alamar blue. FIG. 6 is a set of graphs and images showing that the vaccine synergies with SMC to cause cancer cell death. FIG. 26A shows data from experiments in which EMT6 cells were treated with vehicle or 5 μM LCL 161 (SMC) and 1000 CFU / mL BCG or 1 ng / mL TNFα for 48 hours and viability assessed by Alamar Blue It is a graph. FIG. 6 is a set of graphs and images showing that the vaccine synergies with SMC to cause cancer cell death. Figure 26B intraperitoneally into tumor LCL161 (SMC) and PBS vehicle or 50 mg / kg (it), to it with ^{BCG (1 × 10 5 CFU)} , or ^{BCG (1 × 10 5 CFU)} (ip A set of representative IVIS images (EMT6-Fluc) showing survival of mice bearing mammary fat pad tumors, treated twice) and subjected to live tumor bioluminescence imaging with an IVIS CCD camera at various time points. Scale: p / sec / cm2 / sr. FIG. 7 is a set of paired graphs and images showing that SMC synergizes with type I IFN to cause mammary tumor regression. FIG. 27A: Mouse mammary fat pad injected with EMT6-Fluc tumor, 8 days after implantation orally with vehicle or 50 mg / kg of LCL 161 (SMC) in combination with bovine serum albumin (BSA) Figure 10 is a pair of graphs showing data from experiments treated intraperitoneally (ip) in combination with 1 μg IFNα, or intratumorally (it) in combination with 2 μg IFNα. Left panel shows tumor growth. The right panel represents Kaplan-Meier curves showing mouse survival. Error bars, mean ± s. E. M. FIG. 7 is a set of paired graphs and images showing that SMC synergizes with type I IFN to cause mammary tumor regression. FIG. 27B is a series of representative IVIS images derived from the experiment described in FIG. 27A. Scale: p / sec / cm2 / sr. FIG. 16 is a graph showing that VSV-IFNβ or VSV synergizes with SMC to cause cancer cell death. FIG. 28A shows data from experiments in which EMT6 cells were co-treated with vehicle or 5 μM LCL 161 (SMC) and VSV Δ51-GFP, VSV-IFNβ, or VSV-NIS-IFNβ at different multiplicity of infection (MOI) . Cell viability was assessed 48 hours after treatment with alamar blue. FIG. 16 is a graph showing that VSV-IFNβ or VSV synergizes with SMC to cause cancer cell death. FIG. 28B shows EMT 6 mammary tumor bearing mice treated twice orally with vehicle or 50 mg / kg LCL 161 (SMC) and twice intratumorally with PBS or 1 × 10 ⁸ PFU of VSV-IFNβ-NIS. It is a pair of graphs. FIG. 16 is a graph showing that VSV-IFNβ or VSV synergizes with SMC to cause cancer cell death. FIG. 28C is a pair of graphs when EMT 6 breast tumor bearing mice were treated twice intratumorally with vehicle or 50 mg / kg LCL 161 (SMC) orally and with 1 × 10 ⁸ PFU of VSV. Non-viral and viral triggers are graphs showing that they induce robust expression of TNFα in vivo. Mice were treated with 50 mg poly (I: C) intraperitoneally or with an intravenous injection of 5 × 10 ⁸ PFU of VSV Δ51, VSV-mIFNβ, or Maraba-MG1. At the indicated times, serum was isolated and processed for ELISA to quantify levels of TNFα. FIG. 6 is a set of graphs and images showing that proinflammatory cytokines expressed by the virus synergize with SMC to induce mammary tumor regression. FIG. 30A shows injection of EMT6-Fluc tumor into mouse mammary fat pad, 7 days after implantation orally 1 × 10 ^{8 of} vehicle or 50 mg / kg of LCL 161 (SMC) in combination with PBS FIG. 16 is a pair of graphs showing data from experiments treated with PFU in combination with VSVΔ51-memTNFα (iv), or in combination with 1 × 10 ⁸ PFU in combination with VSVΔ51-solTNFα (iv). Left panel shows tumor growth. The right panel represents Kaplan-Meier curves showing mouse survival. Error bars, mean ± sem. FIG. 6 is a set of graphs and images showing that proinflammatory cytokines expressed by the virus synergize with SMC to induce mammary tumor regression. FIG. 30B is a set of representative bioluminescent IVIS images obtained from the experiment described in FIG. 30A. Scale: p / sec / cm2 / sr. FIG. 6 is a set of graphs and images showing that proinflammatory cytokines expressed by the virus synergize with SMC to induce mammary tumor regression. FIG. 30C shows that mice were injected subcutaneously with CT-26 tumor and 10 days after implantation orally with vehicle or 50 mg / kg of LCL 161 in combination with PBS or 1 × 10 ⁸ PFU of VSV Δ51-solTNFα FIG. 7 is a pair of graphs showing data from experiments treated intratumorally in combination with Left panel shows tumor growth. The right panel represents Kaplan-Meier curves showing mouse survival. Error bars, mean ± sem. SMC is a set of images showing that SMC treatment results in downregulation of cIAP1 / 2 protein in vivo in an orthotopic, syngeneic mouse model of glioblastoma. FIG. 31A shows CT-2A cells implanted intracranially, treated orally with 50 mg / kg LCL 161 (SMC), resected tumors at indicated time points, cIAP1 / 2, XIAP, and β-tubulin FIG. 16 is an image showing an immunoblot derived from an experiment processed for Western blotting using an antibody to SMC is a set of images showing that SMC treatment results in downregulation of cIAP1 / 2 protein in vivo in an orthotopic, syngeneic mouse model of glioblastoma. FIG. 31B shows that CT-2A cells are implanted intracranially, treated intratumorally with 10 μL of 100 μM LCL 161, resected at indicated time points, using antibodies against cIAP1 / 2, XIAP, and β-tubulin 6 is an image showing an immunoblot derived from an experiment processed for Western blotting. FIG. 6 is a set of graphs and images showing that transient proinflammatory responses in the brain synergize with SMC to cause glioblastoma cell death. FIG. 32A is an ELISA to determine the level of soluble TNFα from 300 mg of crude brain protein extract from mice injected intraperitoneally for 12 or 24 hours with PBS or 50 mg of poly (I: C). Is a graph showing data derived from the experiment performed. Brain protein extracts were obtained by mechanical homogenization in saline solution. FIG. 6 is a set of graphs and images showing that transient proinflammatory responses in the brain synergize with SMC to cause glioblastoma cell death. FIG. 32B shows data from alamar blue viability assay of mouse glioblastoma cells (CT-2A, K1580) treated with 70 mg crude brain homogenate and 5 μM LCL 161 (SMC) in culture for 48 hours Is a graph showing Brain homogenates were obtained from mice treated for 12 h with ip injection of poly (I: C) or intravenous injection of 5 × 10 ⁸ PFU of VSV Δ51 or VSV-mIFNβ. FIG. 6 is a set of graphs and images showing that transient proinflammatory responses in the brain synergize with SMC to cause glioblastoma cell death. FIG. 32C depicts a Kaplan-Meier curve showing survival of mice receiving three intracranial treatments of 50 mg poly (I: C). Treatment was performed on day 0, 3 and 7. FIG. 6 is a set of graphs and images showing that transient proinflammatory responses in the brain synergize with SMC to cause glioblastoma cell death. FIG. 32D depicts a Kaplan-Meier curve showing survival of mice bearing CT-2A intracranial tumors that received a combination of SMC, VSVΔ51 or poly (I: C). Mice were treated three times with vehicle, three treatments with 75 mg / kg LCL 161 (oral), three treatments with 5 × 10 ⁸ PFU of VSV Δ51 (iv), or 50 mg poly (I: C) Intracranial, ic) received a combination of 2 treatments. The mice were treated 7, 10 and 14 days after tumor cell implantation with different conditions, except for the poly (I: C) treatment group which received ic injection on 7 and 15 days. The numbers in parentheses indicate the number of mice per group. FIG. 6 is a set of graphs and images showing that transient proinflammatory responses in the brain synergize with SMC to cause glioblastoma cell death. Figure 32E is a series of representative MRI images of a mouse skull derived from the experiment shown in Figure 32D, showing an animal at the endpoint and a representative mouse of the indicated group at 50 days after implantation . The dashed line indicates a brain tumor. FIG. 5 is a graph showing that SMC synergizes with type I IFN to eradicate brain tumors. This graph represents Kaplan-Meier curves showing survival of mice bearing CT-2A that received an intracranial injection of vehicle or 100 μM LCL 161 (SMC) with PBS or 1 μg IFNα at 7 days after implantation. . An overview of the NF-κB signaling pathway. Upon ligand encounter with TNF family receptors, the classical or alternative pathway is activated depending on the activity of cIAP1 / 2. In classical NF-κB activation, RIP1 receives cIAP1 / 2 to K63 ubiquitin binding to form a signaling complex, and after activation of IκB-kinase (IKK), an inhibitor of BB (IκB) Allows phosphorylation. The phosphorylated IκB is degraded releasing the p50 / p65 heterodimer. An alternative pathway is kept inactive by cIAP1 / 2 K48-linked ubiquitination of NF-κB induced kinase (NIK). If NIK is stable, it allows IKK and downstream p100 phosphorylation, resulting in the processing of p100 to p52. This pathway terminates in NF--B heterodimers, translocates to the nucleus, acts as a transcription factor, and regulates expression of target genes. It is described that the process of combining SMC and a monoclonal antibody to PD-1 delayed disease progression and prolonged survival in the mouse MM model. FIG. 35A shows images of mice bearing MPC-11 Fluc cells treated three times weekly with 250 μg of ICI and 50 mg / kg for 2 weeks. Mice were treated with SMC and monoclonal antibodies to PD-1 or CTLA-4. Mice treated with a combination of anti-PD-1 and SMC showed little tumor burden as determined by IVIS bioluminescence imaging of cancer burden versus days after cell implantation. It is described that the process of combining SMC and a monoclonal antibody to PD-1 delayed disease progression and prolonged survival in the mouse MM model. FIG. 35B shows treatment regimens with anti-PD-1, anti-CTLA-4 and SMC. It is described that the process of combining SMC and a monoclonal antibody to PD-1 delayed disease progression and prolonged survival in the mouse MM model. FIG. 35C is a graph showing the number of days of mice surviving after implantation of MPC-11 Fluc cells, as shown in the Kaplan-Meier curve. FIG. 7 is a series of graphs showing that innate immune stimulators synergize with SMC to cause MM cell death. FIG. 36A is a series of bar graphs showing the viability of human cell lines U266, MM1R, and MM1S treated with 1 U / μL of IFNα, IFNβ, and IFNγ in the presence of vehicle or 5 μM SMC. Viability was determined by trypan blue exclusion 24 hours later. FIG. 7 is a series of graphs showing that innate immune stimulators synergize with SMC to cause MM cell death. FIG. 36B is a graph showing the viability of mouse MM cell line MPC-11 treated with 5 μM SMC and VSV Δ51 at various multiplicity of infection (MOI). Viability was assessed 24 hours later using Alamar Blue. FIG. 7 is a series of graphs showing that innate immune stimulators synergize with SMC to cause MM cell death. FIG. 36C is a graph showing the viability of the murine MM cell line MPC-11 treated with 5 μM SMC and VSV mIFN at different multiplicity of infection (MOI). Viability was assessed 24 hours later using Alamar Blue. FIG. 16 shows that IFN and SMC synergize to delay MM disease progression in mice. Mice bearing MPC-11 Fluc cells were treated three times with 1 μg of recombinant IFNα and 50 mg / kg of SMC. FIG. 37A is a series of IVIS bioluminescence images of cancer burden taken at indicated days after MM cell implantation. FIG. 16 shows that IFN and SMC synergize to delay MM disease progression in mice. Mice bearing MPC-11 Fluc cells were treated three times with 1 μg of recombinant IFNα and 50 mg / kg of SMC. FIG. 37B is a Kaplan-Meier curve showing survival time. FIG. 16 shows that IFN and SMC synergize to delay MM disease progression in mice. Mice bearing MPC-11 Fluc cells were treated three times with 1 μg of recombinant IFNα and 50 mg / kg of SMC. FIG. 37C is a diagram showing a treatment regimen. FIG. 8 shows that oncolytic viruses can delay MM disease progression and increase survival. FIG. 38A is an IVIS bioluminescence image taken at indicated days after implantation of mice bearing MPC-11 Fluc cells treated 4 times with 5 × 10 ⁸ pfu of VSV Δ51 and 50 mg / kg of SMC . FIG. 8 shows that oncolytic viruses can delay MM disease progression and increase survival. FIG. 38B is a Kaplan-Meier curve showing survival time. FIG. 8 shows that oncolytic viruses can delay MM disease progression and increase survival. Figure 38C shows a treatment regimen. FIG. 8 shows that glucocorticoid receptor ligands synergize with SMC to sensitize resistant cell lines to SMC-mediated cell death. FIG. 39A is a diagram showing that proteins were extracted from MM1R and MM1S cells for western blotting and equal amounts of protein were used. FIG. 8 shows that glucocorticoid receptor ligands synergize with SMC to sensitize resistant cell lines to SMC-mediated cell death. FIG. 39B shows that cells were treated with 5 μM SMC, 10 μM Dex and 10 μM RU 486 for the indicated times, dead cells were determined as YOYO-1 positive using cell impermeable DNA binding dye, It is a graph which shows having normalized to the confluence of the cell in a well. FIG. 8 shows that glucocorticoid receptor ligands synergize with SMC to sensitize resistant cell lines to SMC-mediated cell death. FIG. 39C shows that cells were treated with 5 μM SMC, 10 μM Dex and 10 μM RU 486 for the indicated times and dead cells were determined as YOYO-1 positive using cell impermeable DNA binding dye, It is a graph which shows having normalized to the confluence of the cell in a well. FIG. 6 shows that SMCs increase NF-κB signaling and cause apoptosis. Human MM cell lines MM1R and MM1S were harvested 1, 16 or 48 hours after treatment with 5 μM SMC. FIG. 40A shows a Western blot for various components of the NF-κB pathway. FIG. 6 shows that SMCs increase NF-κB signaling and cause apoptosis. Human MM cell lines MM1R and MM1S were harvested 1, 16 or 48 hours after treatment with 5 μM SMC. FIG. 40B is a quantification of the band from FIG. 40A, expressed as the ratio of p-p65 to p65, normalized to the untreated control. FIG. 6 shows that SMCs increase NF-κB signaling and cause apoptosis. Human MM cell lines MM1R and MM1S were harvested 1, 16 or 48 hours after treatment with 5 μM SMC. FIG. 40C is a quantification of the bands from FIG. 40A, expressed as the ratio of p-p65 to p52: p100 normalized to untreated controls. FIG. 5 shows that combined treatment of SMC and IFNβ increases NF-κB activity and causes apoptosis. Human cell lines U266, MM1R and MM1S as well as mouse cell lines MPC-11 and Fluc tagged subcell lines were treated with 5 μM SMC and 1 U / μL IFNβ for 1 or 16 hours. Cell pellets were harvested and lysates loaded equally for western blotting. FIG. 8 shows that oncolytic virus in combination with SMC activates NF-κB signaling leading to apoptosis in mouse MM cells. MPC-11 cells were treated with VSVΔ51 or VSVmIFN for 1, 12, or 24 hours. FIG. 42A is a Western blot showing that cell pellets were harvested and lysates were equally loaded for western blotting. FIG. 8 shows that oncolytic virus in combination with SMC activates NF-κB signaling leading to apoptosis in mouse MM cells. MPC-11 cells were treated with VSVΔ51 or VSVmIFN for 1, 12, or 24 hours. FIG. 42B is the protein level quantified from the bands in FIG. 42A and is expressed as the ratio of phospho-p65 to p65. FIG. 8 shows that oncolytic virus in combination with SMC activates NF-κB signaling leading to apoptosis in mouse MM cells. MPC-11 cells were treated with VSVΔ51 or VSVmIFN for 1, 12, or 24 hours. FIG. 42C is the protein level quantified from the bands in FIG. 42A and is expressed as the ratio of p52 to p100. PD-L1 and PD-L2 expression is increased in human MM cell lines after treatment with IFN beta. Expression of PD-L1 and PD-L2 mRNA increases at 6, 12 and 24 hours after IFNβ treatment or treatment with IFNβ and SMC as compared to untreated controls. FIG. 6 is a graph showing that the combination of SMC and immunomodulatory agent results in cancer cell death that also includes CD8 + T cells. FIG. 44A is a graph showing data from experiments in which EMT6 cells were reinjected into the mammary fat pad of double-treated cured mice (180 days from the date after first implantation). FIG. 6 is a graph showing that the combination of SMC and immunomodulatory agent results in cancer cell death that also includes CD8 + T cells. FIG. 44B is a graph showing data from experiments in which CT-2A cells were reinjected into the skull of double-treated cured mice (190 days after the first implantation) experiments. FIG. 6 is a graph showing that the combination of SMC and immunomodulatory agent results in cancer cell death that also includes CD8 + T cells. FIG. 44C shows data from experiments in which CT-2A glioma or EMT6 breast cancer cells were trypsinized, surface stained with conjugated isotype control IgG or anti-PD-L1, and processed for flow cytometry. FIG. FIG. 6 is a graph showing that the combination of SMC and immunomodulatory agent results in cancer cell death that also includes CD8 + T cells. FIG. 44D shows that CD8 + T cells are enriched from splenocytes (derived from naive mice or mice to which EMT6 tumors have previously been cured) using CD8 T cell positive magnetic selection kit to detect IFNγ and granzyme B Is a graph showing data from experiments that were subjected to the ELISpot assay. CD8 + T cells were cocultured with culture medium or cancer cells (the ratio of cancer cells to CD8 + T cells 12: 1) and 10 mg of control IgG or anti-PD-1 for 48 hours. Three mice were used as independent biological replicates (previously EMT6 tumors have been cured). Since 4T1 and EMT6 cells carry the same major histocompatibility antigens, 4T1 cells serve as negative controls. FIG. 5 is a graph showing that SMC synergizes with immune checkpoint inhibitors in an orthotopic mouse model of cancer. FIG. 45A shows that EMT 6 mammary tumor bearing mice are treated intratumorally with PBS or 1 × 10 ⁸ PFU of VSVD 51 once, and 5 days later mice are orally treated with vehicle or 50 mg / kg LCL 161 (SMC) And 250 mg of anti-PD in combination with intraperitoneal (ip) treatment. FIG. 5 is a graph showing that SMC synergizes with immune checkpoint inhibitors in an orthotopic mouse model of cancer. Figure 45B: Data from mice bearing intracranial CT-2A tumors treated 4 times with vehicle or 75 mg / kg LCL 161 (oral) and 250 mg (ip) control IgG, anti-PD-1 or anti-CTLA-4 Is a graph showing FIG. 5 is a graph showing that SMC synergizes with immune checkpoint inhibitors in an orthotopic mouse model of cancer. Figure 45B shows data from mice bearing intracranial GL261 tumors treated 4 times with vehicle or 75 mg / kg LCL 161 (oral) and 250 mg (ip) control IgG, anti-PD-1 or anti-CTLA-4 It is a graph. FIG. 5 is a graph showing that SMC synergizes with immune checkpoint inhibitors in an orthotopic mouse model of cancer. FIG. 16 is a graph showing data of athymic CD-1 nude mice bearing CT-2A intracranial tumors treated with 75 mg / kg LCL 161 (oral) and 250 mg (ip) anti-PD-1. FIG. 5 is a graph showing that SMCs induce the killing of glioblastoma cells in the presence of cytokines or oncolytic viruses. Ara or human (M059K, SNB75, U118) and mouse (CT-2A, GL261) glioblastoma cells treated with vehicle or 5 μM LCL 161 (SMC) and 0.1 ng / mL TNF-α or 0.01 MOI VSV Δ51 for 48 hours Marblue Viability Assay (FIG. 46A). Error bar, average, sd. n = 4. FIG. 46A shows representative data from three independent experiments using biological replicates. Statistical significance was compared to vehicle and BSA treatment using ANOVA using Dunnett's multiple comparison test. If p <0.0001 ( ^* ), then significance is reported. FIG. 5 is a graph showing that SMCs induce the killing of glioblastoma cells in the presence of cytokines or oncolytic viruses. The indicated primary mouse NF1-/ + p53-/ + cell lines were used as vehicle or 5 μM LCL 161 (SMC) and 0.01% BSA, 1 ng / mL TNF-α or non-diffusing VSV Δ51 (VSV Δ51 ΔG of indicated MOI) ) And viability was assessed by Alamar Blue (FIG. 46B). Error bar, average, sd. n = 4. FIG. 46B shows representative data from three independent experiments using biological replicates. Statistical significance was compared to vehicle and BSA treatment using ANOVA using Dunnett's multiple comparison test. If p <0.0001 ( ^* ), then significance is reported. FIG. 5 is a graph showing that SMCs induce the killing of glioblastoma cells in the presence of cytokines or oncolytic viruses. Alamar Blue Viability Assay of human brain tumor initiating cells (BTIC) treated with vehicle or VSV Δ51 or Maraba-MG1 at 5 μM LCL 161 and 0.001 MOI for 48 hours (FIG. 46C). Error bar, average, sd. n = 3. Statistical significance was compared to vehicle and BSA treatment using ANOVA using Dunnett's multiple comparison test. If p <0.0001 ( ^* ), then significance is reported. FIG. 6 is a graph showing that SMC strongly synergizes with TNF-α to induce the death of glioblastoma cells. Viability of mouse glioblastoma CT-2A cells for 48 hours of treatment with 0.01% BSA or 0.1 ng / mL TNF-α and vehicle or 5 μM of the indicated monomer or dimer. Viability was assessed by Alamar Blue. Error bar, average, sd. n = 4. Representative data from two independent experiments using biological replicates. Statistical significance was compared to vehicle and BSA treatment using ANOVA using Dunnett's multiple comparison test. If p <0.0001 ( ^* ), then significance is reported. FIG. 7 is a series of graphs and images showing that resistance to SMC-based combinations in glioblastoma cells is avoided using downregulation of cFLIP. Primary mouse NF1-/ + p53-/ + (K5001) or human (SF 539) glioblastoma cells or human non-transformed cells (GM38) after transfecting non-targeted (NT) or cFLIP siRNA for 48 hours, vehicle Or treated with 5 μM LCL 161 (SMC) and BSA, 0.1 ng / mL TNF-α or non-diffusive VSV Δ51 (VSV Δ51 ΔG; FIG. 48A) of the indicated MOI for 48 hours. Viability was determined by Alamar Blue. Error bar, average, sd. n = 4. Representative data from three independent experiments using biological replicates. Statistical significance was compared to vehicle and BSA treatment using ANOVA using Dunnett's multiple comparison test. If p <0.0001 ( ^* ), then significance is reported. Efficacy of NT siRNA or cFLIP targeting siRNA derived from the experiment in (FIG. 48B). FIG. 7 is a series of graphs and images showing that resistance to SMC-based combinations in glioblastoma cells is avoided using downregulation of cFLIP. Primary mouse NF1-/ + p53-/ + (K5001) or human (SF 539) glioblastoma cells or human non-transformed cells (GM38) after transfecting non-targeted (NT) or cFLIP siRNA for 48 hours, vehicle Or treated with 5 μM LCL 161 (SMC) and BSA, 0.1 ng / mL TNF-α or non-diffusive VSV Δ51 (VSV Δ51 ΔG; FIG. 48A) of the indicated MOI for 48 hours. Viability was determined by Alamar Blue. Error bar, average, sd. n = 4. Representative data from three independent experiments using biological replicates. Statistical significance was compared to vehicle and BSA treatment using ANOVA using Dunnett's multiple comparison test. If p <0.0001 ( ^* ), then significance is reported. Efficacy of NT siRNA or cFLIP targeting siRNA derived from the experiment in (FIG. 48B). FIG. 10 is an image showing the establishment of a mouse syngeneic orthotopic model of glioblastoma. MRI images of C57BL / 6 mice sacrificed intracranially injected with PBS or 5 × 10 ⁴ CT-2A cells and sacrificed at day 35 after implantation are shown. Scale bar, 2 mm. The ruler is shown in cm with the mm section. FIG. 10 is an image showing the establishment of a mouse syngeneic orthotopic model of glioblastoma. Macroscopic images of C57BL / 6 mice sacrificed intracranially injected with PBS or 5 × 10 ⁴ CT-2A cells and sacrificed at 35 days post implantation are shown. Scale bar, 2 mm. The ruler is shown in cm with the mm section. FIG. 5 is a graph showing that SMC synergizes with an innate immune stimulator for treatment of glioblastoma. Scale bar, 2 mm. Alamar Blue Viability Assay of CT-2A cells treated with vehicle or 5 μM LCL 161 and 0.01% BSA or 1 μg / mL IFN-α B / D. Error bar, average, s. D. n = 4 (FIG. 50A). FIG. 5 is a graph showing that SMC synergizes with an innate immune stimulator for treatment of glioblastoma. Scale bar, 2 mm. Mice bearing 7 day old intracranial CT-2A tumors were treated with a combination of 75 mg / kg LCL 161 (oral) and BSA or 1 μg IFN-α B / D (ip) (FIG. 50B). FIG. 50B shows data representing a Kaplan-Meier curve showing mouse survival. Log rank with Holm-Sidak multiple comparisons: ^** , p <0.01; ^*** , p <0.001. The numbers in parentheses represent the number of mice per group. FIG. 16 is an image showing that SMC treatment does not induce downregulation of IAP in brain tissue from non-tumor bearing mice. Mice were treated with 75 mg / kg of LCL 161 (SMC) for the indicated time, and tissues were processed for Western blot using the indicated antibodies. N = 2 for each time point. FIG. 6 is a graph showing that SMC-based combination treatment results in long-term immunological anti-tumor memory. Treat CT-2A cells with vehicle or 5 μM LCL 161 (SMC) and 0.01% BSA, 1 ng / mL TNF-α, 250 U / mL IFN-β or 0.1 MOI VSV Δ51 for 24 hours, using live cells (Zombie Green) Negative) was analyzed by flow cytometry using the indicated antibodies (FIG. 52A). Representative data from three independent experiments using biological replicates. FIG. 6 is a graph showing that SMC-based combination treatment results in long-term immunological anti-tumor memory. EMT6 or breast adenocarcinoma 4T1 cells in mice previously cured using SMC-based treatment of naive mice or mammary fat pad EMT6 (mammary adenocarcinoma, FIG. 52B) or intracranial CT-2A (glioblastoma, FIG. 52C) tumors. Was reinjected into the mammary fat, or subcutaneously (sc) or CT-2A cells intracranially (ic). Cells were implanted 180 days after the first implantation. Data represent Kaplan-Meier curves showing mouse survival. Log rank (compared to the embedding method) using Form-Sidak multiple comparisons: ^* , p <0.05; ^** , p <0.01; ^*** , p <0.001. The numbers in parentheses represent the number of mice per group. FIG. 6 is a graph showing that SMC-based combination treatment results in long-term immunological anti-tumor memory. EMT6 or breast adenocarcinoma 4T1 cells in mice previously cured using SMC-based treatment of naive mice or mammary fat pad EMT6 (mammary adenocarcinoma, FIG. 52B) or intracranial CT-2A (glioblastoma, FIG. 52C) tumors. Was reinjected into the mammary fat, or subcutaneously (sc) or CT-2A cells intracranially (ic). Cells were implanted 180 days after the first implantation. Data represent Kaplan-Meier curves showing mouse survival. Log rank (compared to the embedding method) using Form-Sidak multiple comparisons: ^* , p <0.05; ^** , p <0.01; ^*** , p <0.001. The numbers in parentheses represent the number of mice per group. FIG. 5 is a graph showing that SMC treatment does not abolish the expression of checkpoint inhibitor molecules or MHC I / II proteins. Treating SNB75 cells with vehicle or 5 μM LCL 161 (SMC) and 1 ng / mL TNF-α, 250 U / mL IFN-β or 0.1 MOI VSV Δ51 for 24 hours and showing live cells (Zombie Green negative), Was processed for flow cytometry using the antibody. Representative data from three independent experiments. FIG. 16 is a graph showing that SMC synergizes with antibodies that target immune checkpoints in a mouse model of glioblastoma. Splenic CD8 + T cells were enriched from naive or CT-2A tumor-healed mice and subjected to ELISpot assay for detection of IFN-γ and GrzB. Cancer cells (CT-2A, LLC) were cocultured with CD8 + cells (25: 1 ratio) and 10 μg / mL of control IgG or α-PD-1 for 48 hours. n = 4 mice / group (FIG. 54A). Significance was compared to naive CD8 + T cells co-incubated with CT-2A cells as assessed by ANOVA using Dunnett's multiple comparison test. ^* , P <0.05; ^** , p <0.01; ^*** , p <0.001. In FIG. 54A, crosses indicate mean, solid horizontal lines indicate median, box indicates first to third quartiles, and whiskers indicate minimum-maximum range of values. FIG. 16 is a graph showing that SMC synergizes with antibodies that target immune checkpoints in a mouse model of glioblastoma. Mice bearing intracranial CT-2A tumors were treated orally (SMC) with 75 mg / kg LCL 161 at days 14, 16, 21 and 23 after implantation (FIG. 54B). In FIG. 54B, crosses indicate mean, solid horizontal lines indicate median, box indicates first to third quartile, and whiskers indicate minimum-maximum range of values. FIG. 16 is a graph showing that SMC synergizes with antibodies that target immune checkpoints in a mouse model of glioblastoma. Living cells derived from tumor masses were analyzed by flow cytometry for detection of CD45 (BV605), CD3 (APC-Cy7), CD8 (PE) and PD-1 (BV421). Experimentally derived live tumor cells were analyzed by flow cytometry using antibodies CD45 (PE) and PD-L1 (BV421) (FIG. 54C). n = 6 mice / group. FMO, fluorescence -1. Statistical significance was assessed by t-test. In FIG. 54C, crosses indicate mean, solid horizontal lines indicate median, box indicates first to third quartiles, and whiskers indicate minimum-maximum range of values. FIG. 16 is a graph showing that SMC synergizes with antibodies that target immune checkpoints in a mouse model of glioblastoma. Mice bearing an intracranial CT-2A (Figure 54D, Figure 54F and Figure 54G) or GL261 (Figure 54E) tumor are orally administered with vehicle, 75 mg / kg LCL 161, at the indicated times (Figure 54D, Figure 54E). And Figure 54G) or with vehicle or 30 mg / kg birinapant intraperitoneally (ip; Figure 54F) and with a combination of 250 μg of IgG, α-PD-1 or α-CTLA-4 (ip), or Treatment with both combinations (FIG. 54G). Data represent Kaplan-Meier curves showing mouse survival. Log rank with Holm-Sidak multiple comparisons: ^* , p <0.05; ^** , p <0.01; ^*** , p <0.001. The numbers in parentheses represent the number of mice per group. FIG. 54D shows representative data from two independent experiments. FIG. 16 is a graph showing that SMC synergizes with antibodies that target immune checkpoints in a mouse model of glioblastoma. Mice bearing an intracranial CT-2A (Figure 54D, Figure 54F and Figure 54G) or GL261 (Figure 54E) tumor are orally administered with vehicle, 75 mg / kg LCL 161, at the indicated times (Figure 54D, Figure 54E). And Figure 54G) or with vehicle or 30 mg / kg birinapant intraperitoneally (ip; Figure 54F) and with a combination of 250 μg of IgG, α-PD-1 or α-CTLA-4 (ip), or Treatment with both combinations (FIG. 54G). Data represent Kaplan-Meier curves showing mouse survival. Log rank with Holm-Sidak multiple comparisons: ^* , p <0.05; ^** , p <0.01; ^*** , p <0.001. The numbers in parentheses represent the number of mice per group. FIG. 16 is a graph showing that SMC synergizes with antibodies that target immune checkpoints in a mouse model of glioblastoma. Mice bearing an intracranial CT-2A (Figure 54D, Figure 54F and Figure 54G) or GL261 (Figure 54E) tumor are orally administered with vehicle, 75 mg / kg LCL 161, at the indicated times (Figure 54D, Figure 54E). And Figure 54G) or with vehicle or 30 mg / kg birinapant intraperitoneally (ip; Figure 54F) and with a combination of 250 μg of IgG, α-PD-1 or α-CTLA-4 (ip), or Treatment with both combinations (FIG. 54G). Data represent Kaplan-Meier curves showing mouse survival. Log rank with Holm-Sidak multiple comparisons: ^* , p <0.05; ^** , p <0.01; ^*** , p <0.001. The numbers in parentheses represent the number of mice per group. FIG. 16 is a graph showing that SMC synergizes with antibodies that target immune checkpoints in a mouse model of glioblastoma. Mice bearing an intracranial CT-2A (Figure 54D, Figure 54F and Figure 54G) or GL261 (Figure 54E) tumor are orally administered with vehicle, 75 mg / kg LCL 161, at the indicated times (Figure 54D, Figure 54E). And Figure 54G) or with vehicle or 30 mg / kg birinapant intraperitoneally (ip; Figure 54F) and with a combination of 250 μg of IgG, α-PD-1 or α-CTLA-4 (ip), or Treatment with both combinations (FIG. 54G). Data represent Kaplan-Meier curves showing mouse survival. Log rank with Holm-Sidak multiple comparisons: ^* , p <0.05; ^** , p <0.01; ^*** , p <0.001. The numbers in parentheses represent the number of mice per group. FIG. 6 is a series of graphs showing that SMC treatment results in the upregulation of PD-1 in CD8 T cells. Mice bearing intracranial CT-2A tumors were treated orally (SMC) with 75 mg / kg of LCL 161 at days 14, 16, 21 and 23 after implantation. Living cells derived from CT-2A tumors were processed for flow cytometry using antibodies CD45 (BV605), CD3 (APC-Cy7), CD8 (PE), and PD-1 (BV421). FIG. 16 is a graph showing that SMC synergizes with immune checkpoint inhibitors for the treatment of mouse models of multiple myeloma. MPC-11 cells were treated with vehicle or 5 μM LCL 161 (SMC) and 0.1 ng / mL TNF-α, 250 U / mL IFN-α, or 250 U / mL IFN-β (FIG. 56A). Viability was determined by Alamar Blue 48 hours after treatment. Error bar, average, sd. n = 4. Statistical significance was compared to vehicle and BSA treatment using ANOVA using Dunnett's multiple comparison test. If p <0.0001 ( ^*** ), then significance is reported. Representative data from three independent experiments using biological replicates. FIG. 16 is a graph showing that SMC synergizes with immune checkpoint inhibitors for the treatment of mouse models of multiple myeloma. MPC-11 cells were dissociated and processed for flow cytometry using PE-Cy7 conjugated isotype IgG or PD-L1 (FIG. 56B). FIG. 16 is a graph showing the combination of SMC and an antibody that targets an immune checkpoint inhibitor in a mouse model of breast cancer. Viability assay of EMT6 cells treated with vehicle or 5 μM LCL 161 (SMC) and 0.1 ng / mL TNF-α, 250 U / mL IFN-β or 0.1 MOI VSV Δ51 for 48 hours (FIG. 57A). Error bar, average, sd. n = 4. Statistical significance was compared to vehicle and BSA treatment using ANOVA using Dunnett's multiple comparison test. If p <0.0001 ( ^*** ), then significance is reported. Representative data from three independent experiments using biological replicates. FIG. 16 is a graph showing the combination of SMC and an antibody that targets an immune checkpoint inhibitor in a mouse model of breast cancer. EMT6 cells were dissociated and processed for flow cytometry using PE-Cy7 conjugated isotype IgG or PD-L1 (FIG. 57B). Representative data from three independent experiments. FIG. 16 is a graph showing the combination of SMC and an antibody that targets an immune checkpoint inhibitor in a mouse model of breast cancer. Intratumoral treatment of mice bearing approximately 100 mm3 of EMT6-Fluc tumor with PBS or 5 × 10 ⁸ PFU of VSV Δ51 at indicated times after implantation with vehicle or 50 mg / kg LCL 161 (SMC) Treated orally and intraperitoneally with 250 μg of IgG or α-PD-1 (FIG. 57C). Left panel shows tumor growth. Error bar, average, sem. The right panel represents Kaplan-Meier curves showing mouse survival. Log rank with Holm-Sidak multiple comparisons: ^* , p <0.05; ^** , p <0.01. The numbers in parentheses represent the number of mice per group. Figure 6 is a graph showing that inclusion of SMC increases the immune response in the presence of glioblastoma cells. The expression of the indicated factors was shown in CT-2A cells co-incubated for 48 hours with splenocytes from intracranial CT-2A tumors previously cured by naive mice or co-treatment with SMC and anti-PD-1 Detection was by ELISA derived from cell culture supernatant (ratio of CT-2A cells to splenocytes 1:20; FIG. 58A). Crosses indicate means, solid horizontal lines indicate medians, boxes indicate first to third quartiles, and whiskers indicate minimum-maximum range of values. Statistical significance was compared to naive CD8 + T cells as assessed by ANOVA using Dunnett's multiple comparison test. ^* , P <0.05; ^** , p <0.01; ^*** , p <0.001. Figure 6 is a graph showing that inclusion of SMC increases the immune response in the presence of glioblastoma cells. The indicated cytokines were determined by ELISA derived from CT-2A cells cocultured with splenocytes from naive or cured mice and treated for 48 hours with vehicle or 5 μM LCL 161 (SMC) (FIG. 58B) . Crosses indicate means, solid horizontal lines indicate medians, boxes indicate first to third quartiles, and whiskers indicate minimum-maximum range of values. Statistical significance was compared to vehicle and IgG treated T cells as assessed by ANOVA using Dunnett's multiple comparison test. ^** , p <0.01; ^*** , p <0.001. FIG. 8 is an image and a graph showing that CD8 + T cells are required for synergy between SMC and immune checkpoint inhibitors for the treatment of glioblastoma. CT-2A cells co-incubated for 48 hours with splenocytes from intracranial CT-2A tumors previously cured by naive mice or co-treatment with SMC and anti-PD-1 for expression of the indicated immune factors Was detected by ELISA derived from cell culture supernatants (ratio of CT-2A cells to splenocytes 1:20; FIG. 59A). The data is plotted as a heatmap using normalized scaling. Data box and whisker plots are shown in FIG. 58A. FIG. 8 is an image and a graph showing that CD8 + T cells are required for synergy between SMC and immune checkpoint inhibitors for the treatment of glioblastoma. Quantification of the indicated factors is derived from CT-2A cells cocultured with splenocytes from naive or cured mice (1:20 ratio) and treated with vehicle or 5 μM LCL 161 (SMC) for 48 hours Detected by ELISA (FIG. 59B). FIG. 8 is an image and a graph showing that CD8 + T cells are required for synergy between SMC and immune checkpoint inhibitors for the treatment of glioblastoma. Splenocytes from naive or cured mice are co-administered with mKate2-tagged CT-2A cells (CT-2A-mKate2) in the presence of 20 μg / mL of control IgG or anti-PD1 and 5 μM of the indicated SMC. It cultured (FIG. 59C). Counting of CT-2A-mKate2 cells was performed using an Incucyte Zoom. Crosses indicate means, solid horizontal lines indicate medians, boxes indicate first to third quartiles, and whiskers indicate minimum-maximum range of values. Statistical significance was compared to naive splenocytes as assessed by ANOVA using Dunnett's multiple comparison test. ^* Significance is reported if <<0.0001. N = 6 for naive mice and n = 6 for cured mice. Scale bar, 100 μm. FIG. 8 is an image and a graph showing that CD8 + T cells are required for synergy between SMC and immune checkpoint inhibitors for the treatment of glioblastoma. C57BL / 6 mice bearing intracranial CT-2A tumors, on the date indicated, IgG (ip) and vehicle (oral) or α-PD-1 (ip) and 75 mg / kg LCL 161 (oral), and Treated with a combination of ip administration of IgG, α-CD4 or α-CD8 (all antibodies were 250 μg; FIG. 59D). FIG. 8 is an image and a graph showing that CD8 + T cells are required for synergy between SMC and immune checkpoint inhibitors for the treatment of glioblastoma. CD-1 nude mice bearing intracranial CT-2A tumors combined with vehicle or 75 mg / kg LCL 161 (oral) and PBS or 250 μg IgG or α-PD-1 (intraperitoneal) at indicated times Treatment (ip; FIG. 59E). Data represent Kaplan-Meier curves showing mouse survival. Log rank with Holm-Sidak multiple comparisons: ^* , p <0.05; ^** , p <0.01. The numbers in parentheses represent the number of mice per group. FIG. 6 is a series of graphs showing that the combination of SMC and immune checkpoint inhibitor treatment results in an increase in the systemic presence of proinflammatory cytokines. Mouse-derived sera were processed for multiplex ELISA for quantification of the indicated proteins. Crosses indicate means, solid horizontal lines indicate medians, boxes indicate first to third quartiles, and whiskers indicate minimum-maximum range of values. Statistical significance was compared to vehicle and IgG treated mice as assessed by ANOVA using Dunnett's multiple comparison test. ^* , P <0.05. N = 6 for each treatment group. FIG. 5 is a graph showing that treatment with SMCs and immune checkpoint inhibitors in mouse models of glioblastoma results in altered immune effector cell infiltration. Mice bearing intracranial CT-2A tumors were treated orally (SMC) with vehicle or 75 mg / kg LCL 161 and intraperitoneally with 250 μg IgG or anti-PD-1 at indicated times (Figure 61A). The mice were sacrificed 27 days after implantation. All panels: Crosses indicate means, solid horizontal lines indicate medians, boxes indicate first to third quartiles, and whiskers indicate minimum-maximum range of values. Statistical significance was compared to vehicle and IgG treated mice as assessed by ANOVA using Dunnett's multiple comparison test. ^* , P <0.05, ^** , p <0.01. N = 6 for each treatment group. FIG. 5 is a graph showing that treatment with SMCs and immune checkpoint inhibitors in mouse models of glioblastoma results in altered immune effector cell infiltration. Living T cells isolated from tumors were treated with the following antibodies: CD45 (PE-Cy5), CD3 (APC), CD4 (PE-Cy7), CD8 (BV786), CD25 (BV605) and PD-1 (BV421) 61B-61E) were processed for flow cytometry. All panels: Crosses indicate means, solid horizontal lines indicate medians, boxes indicate first to third quartiles, and whiskers indicate minimum-maximum range of values. Statistical significance was compared to vehicle and IgG treated mice as assessed by ANOVA using Dunnett's multiple comparison test. ^* , P <0.05, ^** , p <0.01. N = 6 for each treatment group. FIG. 5 is a graph showing that treatment with SMCs and immune checkpoint inhibitors in mouse models of glioblastoma results in altered immune effector cell infiltration. Living T cells isolated from tumors were treated with the following antibodies: CD45 (PE-Cy5), CD3 (APC), CD4 (PE-Cy7), CD8 (BV786), CD25 (BV605) and PD-1 (BV421) 61B-61E) were processed for flow cytometry. All panels: Crosses indicate means, solid horizontal lines indicate medians, boxes indicate first to third quartiles, and whiskers indicate minimum-maximum range of values. Statistical significance was compared to vehicle and IgG treated mice as assessed by ANOVA using Dunnett's multiple comparison test. ^* , P <0.05, ^** , p <0.01. N = 6 for each treatment group. FIG. 5 is a graph showing that treatment with SMCs and immune checkpoint inhibitors in mouse models of glioblastoma results in altered immune effector cell infiltration. Living T cells isolated from tumors were treated with the following antibodies: CD45 (PE-Cy5), CD3 (APC), CD4 (PE-Cy7), CD8 (BV786), CD25 (BV605) and PD-1 (BV421) 61B-61E) were processed for flow cytometry. All panels: Crosses indicate means, solid horizontal lines indicate medians, boxes indicate first to third quartiles, and whiskers indicate minimum-maximum range of values. Statistical significance was compared to vehicle and IgG treated mice as assessed by ANOVA using Dunnett's multiple comparison test. ^* , P <0.05, ^** , p <0.01. N = 6 for each treatment group. FIG. 5 is a graph showing that treatment with SMCs and immune checkpoint inhibitors in mouse models of glioblastoma results in altered immune effector cell infiltration. Living T cells isolated from tumors were treated with the following antibodies: CD45 (PE-Cy5), CD3 (APC), CD4 (PE-Cy7), CD8 (BV786), CD25 (BV605) and PD-1 (BV421) 61B-61E) were processed for flow cytometry. All panels: Crosses indicate means, solid horizontal lines indicate medians, boxes indicate first to third quartiles, and whiskers indicate minimum-maximum range of values. Statistical significance was compared to vehicle and IgG treated mice as assessed by ANOVA using Dunnett's multiple comparison test. ^* , P <0.05, ^** , p <0.01. N = 6 for each treatment group. FIG. 5 is a graph showing that treatment with SMCs and immune checkpoint inhibitors in mouse models of glioblastoma results in altered immune effector cell infiltration. Live cells derived from the experiment in (a) were treated with the following antibodies: CD45 (BV605), CD11b (APC-Cy7), Gr1 (BV786), F4 / 80 (PE) and CD3 (APC; FIG. 61F and FIG. 61G) Processed for flow cytometry using. All panels: Crosses indicate means, solid horizontal lines indicate medians, boxes indicate first to third quartiles, and whiskers indicate minimum-maximum range of values. Statistical significance was compared to vehicle and IgG treated mice as assessed by ANOVA using Dunnett's multiple comparison test. ^* , P <0.05, ^** , p <0.01. N = 6 for each treatment group. FIG. 5 is a graph showing that treatment with SMCs and immune checkpoint inhibitors in mouse models of glioblastoma results in altered immune effector cell infiltration. Live cells derived from the experiment in (a) were treated with the following antibodies: CD45 (BV605), CD11b (APC-Cy7), Gr1 (BV786), F4 / 80 (PE) and CD3 (APC; FIG. 61F and FIG. 61G) Processed for flow cytometry using. All panels: Crosses indicate means, solid horizontal lines indicate medians, boxes indicate first to third quartiles, and whiskers indicate minimum-maximum range of values. Statistical significance was compared to vehicle and IgG treated mice as assessed by ANOVA using Dunnett's multiple comparison test. ^* , P <0.05, ^** , p <0.01. N = 6 for each treatment group. FIG. 5 is a graph and image showing that the combination of SMC and an immune checkpoint inhibitor induces proinflammatory cytokine responses, and the efficacy is dependent on type I IFN signaling. The living cells derived from brain tumor were isolated and the following antibodies: CD45 (BV605), CD3 (APC-Cy7), Cd4 (PE-Cy7), CD8 (BV786 / 0), IFN-γ (BV421), TNF-α Processed for flow cytometry using (PE) and GrzB (AF 647; FIGS. 62A-D). Crosses indicate means, solid horizontal lines indicate medians, boxes indicate first to third quartiles, and whiskers indicate minimum-maximum range of values. Statistical significance was compared to vehicle and IgG treated mice as assessed by ANOVA using Dunnett's multiple comparison test. ^* , P <0.05. N = 6 for each treatment group. FIG. 5 is a graph and image showing that the combination of SMC and an immune checkpoint inhibitor induces proinflammatory cytokine responses, and the efficacy is dependent on type I IFN signaling. The living cells derived from brain tumor were isolated and the following antibodies: CD45 (BV605), CD3 (APC-Cy7), Cd4 (PE-Cy7), CD8 (BV786 / 0), IFN-γ (BV421), TNF-α Processed for flow cytometry using (PE) and GrzB (AF 647; FIGS. 62A-D). Crosses indicate means, solid horizontal lines indicate medians, boxes indicate first to third quartiles, and whiskers indicate minimum-maximum range of values. Statistical significance was compared to vehicle and IgG treated mice as assessed by ANOVA using Dunnett's multiple comparison test. ^* , P <0.05. N = 6 for each treatment group. FIG. 5 is a graph and image showing that the combination of SMC and an immune checkpoint inhibitor induces proinflammatory cytokine responses, and the efficacy is dependent on type I IFN signaling. The living cells derived from brain tumor were isolated and the following antibodies: CD45 (BV605), CD3 (APC-Cy7), Cd4 (PE-Cy7), CD8 (BV786 / 0), IFN-γ (BV421), TNF-α Processed for flow cytometry using (PE) and GrzB (AF 647; FIGS. 62A-D). Crosses indicate means, solid horizontal lines indicate medians, boxes indicate first to third quartiles, and whiskers indicate minimum-maximum range of values. Statistical significance was compared to vehicle and IgG treated mice as assessed by ANOVA using Dunnett's multiple comparison test. ^* , P <0.05. N = 6 for each treatment group. FIG. 5 is a graph and image showing that the combination of SMC and an immune checkpoint inhibitor induces proinflammatory cytokine responses, and the efficacy is dependent on type I IFN signaling. The living cells derived from brain tumor were isolated and the following antibodies: CD45 (BV605), CD3 (APC-Cy7), Cd4 (PE-Cy7), CD8 (BV786 / 0), IFN-γ (BV421), TNF-α Processed for flow cytometry using (PE) and GrzB (AF 647; FIGS. 62A-D). Crosses indicate means, solid horizontal lines indicate medians, boxes indicate first to third quartiles, and whiskers indicate minimum-maximum range of values. Statistical significance was compared to vehicle and IgG treated mice as assessed by ANOVA using Dunnett's multiple comparison test. ^* , P <0.05. N = 6 for each treatment group. FIG. 5 is a graph and image showing that the combination of SMC and an immune checkpoint inhibitor induces proinflammatory cytokine responses, and the efficacy is dependent on type I IFN signaling. Mouse-derived sera were processed for multiplex ELISA for quantification of the indicated proteins (FIG. 62E). The data is plotted as a heatmap using normalized scaling. N = 6 for each treatment group. FIG. 5 is a graph and image showing that the combination of SMC and an immune checkpoint inhibitor induces proinflammatory cytokine responses, and the efficacy is dependent on type I IFN signaling. Mice were treated and intracranial CT-2A tumors were processed for quantification of 176 cytokine and chemokine genes by RT-qPCR (FIG. 62F). The normalized heatmaps of two major groups identified by hierarchical clustering are shown. N = 4 for each treatment group. FIG. 5 is a graph and image showing that the combination of SMC and an immune checkpoint inhibitor induces proinflammatory cytokine responses, and the efficacy is dependent on type I IFN signaling. Mice bearing intracranial CT-2A tumors are treated with vehicle or 75 mg / kg LCL 161 (oral), or days with the indicated days of implantation, or with the relevant isotype IgG control or 2.5 mg α-IFNAR1, 350 μg α Treated intraperitoneally with IFN-γ or 250 μg of α-PD-1 (FIG. 62G). Significance was compared to vehicle and IgG treated mice as assessed by ANOVA using Dunnett's multiple comparison test. ^* , P <0.05. The numbers in parentheses indicate the size of the treatment group. FIG. 16 is an image showing that proinflammatory cytokines and the chemoattractant chemokine gene signature are upregulated by the combined treatment of SMC and an immune checkpoint inhibitor. Intracranial CT-2A tumors were processed for quantification of 176 cytokine and chemokine genes by RT-qPCR. The normalized heat maps of the major groups identified by hierarchical clustering are shown. N = 4 for each treatment group. FIG. 6 is a series of graphs showing that SMCs enhance clonal expansion of CD8 + T cells in the presence of glioblastoma target cells. Isolated spleen CD8 + T cells from mice in which CT-2A tumors had been previously cured are loaded into CFSE and either vehicle or 5 μM LCL 161 (SMC) or 20 μg / mL control IgG or anti-PD-1 In the presence, they were co-incubated for 96 hours with CT-2A cells (10: 1 ratio). Live cells were processed for flow cytometry. Significance was compared to vehicle and IgG treated mice as assessed by ANOVA using Dunnett's multiple comparison test. ^* , P <0.05; ^** , p <0.01; ^*** , p <0.001. N = 5 for each treatment group. FIG. 8 is a graph and image showing that the proinflammatory cytokine TNF-α is required for T cell mediated cell death of glioblastoma cells upon treatment with Smac mimetics and immune checkpoint inhibitors. Isolated CD8 T cells from spleen and lymph nodes from mice where intracranial CT-2A tumors were previously cured, vehicle or 5 μM LCL 161 and 20 μg / mL isotype matched IgG or α-PD- The cells were cocultured with CT-2A cells for 24 hours in the presence of 1. Live T cells were processed for flow cytometry using the following antibodies: CD3 (APC-Cy7), CD8 (BV711), GrzB (AF647) and TNF-α (PE; FIG. 65A). Crosses indicate means, solid horizontal lines indicate medians, boxes indicate first to third quartiles, and whiskers indicate minimum-maximum range of values. Significance was compared to vehicle and IgG treated mice as assessed by ANOVA using Dunnett's multiple comparison test. ^* , P <0.05; ^** , p <0.01; ^*** , p <0.001. N = 5 for each treatment group. FIG. 8 is a graph and image showing that the proinflammatory cytokine TNF-α is required for T cell mediated cell death of glioblastoma cells upon treatment with Smac mimetics and immune checkpoint inhibitors. MKate2-tagged CT-2A cells (CT-2A-mKate2) for 72 h in the presence of vehicle or 5 μM LCL 161 and 20 μg / mL control IgG, α-PD-1 or α-TNF-α Co-cultured (FIG. 65B). A count of mKate2 positive cells was obtained using Inuccyte Zoom software. Crosses indicate means, solid horizontal lines indicate medians, boxes indicate first to third quartiles, and whiskers indicate minimum-maximum range of values. Significance was compared to vehicle and IgG treated mice as assessed by ANOVA using Dunnett's multiple comparison test. p <0.01; ^*** , p <0.001. N = 5 for each treatment group. Scale bar, 100 μm. FIG. 8 is a graph and image showing that the proinflammatory cytokine TNF-α is required for T cell mediated cell death of glioblastoma cells upon treatment with Smac mimetics and immune checkpoint inhibitors. Mice bearing an intracranial CT-2A tumor, vehicle or 75 mg / kg LCL 161 (oral) at the indicated days after implantation, or a related isotype IgG control or 500 μg α-TNF-α or 250 μg Treatment intraperitoneally with α-PD-1 (FIG. 65C). Significance was compared to vehicle and IgG treated mice as assessed by ANOVA using Dunnett's multiple comparison test. ^** , p <0.01. The numbers in parentheses represent the number of mice per group. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a schematic showing that SMCs are immunomodulators that act on tumors and immune cells to eradicate cancer through the innate and adaptive immune systems. A model is shown that demonstrates the immunomodulatory effects of single agents and combinations of Smac mimetics based on our results. The effects of IAP antagonism on these immune cells or tumor cells are outlined below: (1) SMC stimulate the production of cytokines and chemokines from various immune cells such as macrophages or T cells, and the tumor microenvironment It results in infiltration of immune cells. (2) SMC treatment decreases the immunosuppressive macrophage M2 population and simultaneously increases the proinflammatory M1 population. (3) SMC deplete cIAP1 and cIAP2 to sensitize tumors to cell death by immune ligands such as TNF-α or TRAIL1. Tumor cell death is sensed by the immune system leading to priming of cytotoxic T cell (CTL) responses. (4) SMCs stimulate the TNF / TNFR family member CD40L / CD40 signaling pathway on antigen presenting cells (APCs) to promote dendritic cell (DC) and macrophage differentiation and maturation. APCs present tumor antigens to the immune system and further release cytotoxic inflammatory cytokines. (5) As a result of degradation of cIAP1 and cIAP2 by SMC treatment, SMC activates the alternative NF--B pathway, eliminating the need for TNF superfamily ligands (such as 4-1BB), and thus T cell costimulatory Provide a signal. (6) SMCs have been shown to increase cell death mediated by CTL and natural killer cells. Granzyme B-mediated cell death is blocked by X-linked IAP, XIAP, which blocking can be overcome by mitochondrial release of Smac or by its drug mimic SMC13-15. FIG. 5 is a schematic showing cooperative and complementary mechanisms for the synergism of SMC and immune checkpoint inhibitor (ICI). (1) The presence of therapeutic recombinant antibodies that block the PD-1 / PD-L1 axis is determined by CD8 + using its associated antigen presented by cancer cells via major histocompatibility complex I (MHC-I) molecules. Enables T cell receptor (TCR) signaling of T cells. Simultaneous depletion of IAP by SMC treatment probably enhances T cell activation by providing a tumor necrosis factor receptor superfamily (TNFRSF) costimulatory response (similar to 4-1BB or OX40 activation) Results in enhanced tumor specific CD8 + T cell activation and proliferation. As a result, granzyme B (GrzB) and perforin (Pfn) are secreted and kill the target cells. (2) SMC-mediated antagonism of XIAP, a casp-3 inhibitor, can result in enhanced cell death of tumor cells by GrzB. (3) Depletion of cIAP1 and cIAP2 by SMC results in an increase in the local production of TNF-α by T cells in the tumor microenvironment, which is probably an effect mediated by activation of the alternative ND-κB pathway. (4) As a result of cIAP1 / 2 loss, SMC-treated cancer cells are sensitized to cell death induction in the presence of proinflammatory cytokines such as TNF-α. It is an image showing a full length Western blot. It is an image showing a full length Western blot. It is an image showing a full length Western blot. It is an image showing a full length Western blot.

  The present invention includes methods and compositions for enhancing the efficacy of Smac mimetic compounds (SMCs) in the treatment of cancer. In particular, the invention includes methods and compositions for combination therapy comprising SMC and a second agent that stimulates one or more cell death pathways inhibited by cIAP1 and / or cIAP2. The second agent may be, for example, a TLR agonist, a virus such as a oncolytic virus, or an interferon or related agent.

  The data provided herein demonstrate that treatment with agents and SMC results in tumor regression and permanent cure in vivo (see, eg, Example 1). These combination therapies are well tolerated by mice and body weight returns to pre-treatment levels soon after cessation of therapy. The combination therapy tested was able to treat several treatment refractory aggressive mouse models of cancer. One of ordinary skill in the art, based on the disclosure and data provided herein, any one or more of various SMCs and any one of various agents such as TLR agonists, pathogens, or pathogen mimics. It can be appreciated that one or more can be combined in one or more embodiments of the present invention to promote apoptosis and treat cancer.

  Other approaches have been tried to improve SMC therapy, but it was very rare that a complete response was observed, especially in an aggressive immune response model system. Some embodiments of the present invention may have certain advantages, including the treatment of cancer with a pathogen mimic, eg, a pathogen mimic that has a mechanism of action that is partially dependent on TRAIL. First, this approach can induce TNFα-mediated apoptosis and necrosis: a treatment that simultaneously induces multiple different cell death mechanisms, given the plasticity and heterogeneity of some advanced cancers May have higher efficacy than those that do not. Second, pathogen mimics can elicit an integrated innate immune response, including a layer of negative feedback. These feedback mechanisms act to moderate the cytokine response in a way that is difficult to replicate using recombinant proteins, and thus can act as a safeguard for this combination therapy strategy.

  Multiple myeloma (MM) is an incurable cancer characterized by rapid proliferation of plasma cells in the bone marrow. MM is the second most common hematologic malignancy and its median survival is only 3-5 years after diagnosis. Because MM cells reside in the bone marrow compartment, they cause bone resorption leading to fractures and immunosuppression. MM cells are seeded into other tissues to form plasmacytomas, which may have an aggressive leukemic phase. Current therapies can prolong survival and reduce symptoms, but they are not curative treatments. New therapies are definitely needed to combat treatment resistance and inevitable recurrence.

  Malignant cells depend on the bone marrow microenvironment in the early stages of the disease, in particular on TNF-α and interleukin-6 (IL-6) derived from cells in the bone marrow microenvironment. As the disease progresses, the cells become independent of their environment and survive with the high autocrine production of TNFα. Throughout all stages, cells have high levels of NF-κB signaling that enhance their survival, in part due to common mutations in key components of the pathway. Targeting of the NF-κB pathway in MM has been used in many standard MMs, such as the proteasome inhibitor bortezomib, the immunomodulator (IMiD) thalidomide and lenalidomide and the synthetic glucocorticoid dexamethasone etc. It contributes to the increase of the efficacy of the therapeutic agent.

  TNFα-mediated NF-κB signaling can be switched from survival promoting to apoptotic signaling by removal of cellular inhibitors of apoptosis (cIAP); this process is selective for cancer cells it is conceivable that. cIAP1 and cIAP2 act interchangeably as E3 ligases in all members of the TNFα receptor superfamily to ubiquitinate specific proteins to form a scaffold for signaling complexes or for degradation Target. An example of this can be seen in both arms of the NF-κB pathway: RIP1 is ubiquitinated by K63 binding to form a scaffold signaling complex that is required for activation of the classical pathway, but NIK It accepts K48-linked ubiquitination that targets it for degradation, keeping the alternative pathway inactive (FIG. 35). SMCs are a new class of anti-cancer therapeutics that mimics the endogenous Smac protein involved in the activation of the intrinsic apoptotic pathway. Smac peptides and SMCs bind to the BIR domain of cIAP, causing them to self ubiquitinate and target them for proteasomal degradation. When RIP1 is no longer ubiquitinated, it becomes free to form lipoptosomes, initiating a caspase cascade and cell death.

  SMCs have been shown to potently synergize with TNFα in many cancer lineages to induce NF-κB-mediated apoptosis. SMCs also have synergistic cancer cell killing, along with other proinflammatory cytokines such as IFN, which can be induced by TLR agonists or oncolytic viruses. SMC can further standardize the therapeutic agents used for MM to enhance apoptosis of cancer cells. Several clinical trials currently conducted to evaluate the efficacy of SMC with chemotherapeutic agents in MM as well as other cancers show high therapeutic potential.

  Activation of the immune system increases cytokine production and favors SMC-mediated MM cell killing. However, this cytokine production can have undesirable consequences on MM cells. Many innate immune stimulators, such as IFN and TLR agonists, have been shown to upregulate ligands of the immune checkpoint PD-1. PD-1 is expressed on the surface of T cells and NK cells. When PD-1 binds to its ligands PD-L1 and PD-L2, it acts as a costimulatory signal for the T cell receptor to suppress the cytotoxic potential of T cells. PD-L1 is constitutively expressed at low levels in many tissues and can be upregulated, possibly to prevent autoimmune reactions. However, PD-L1 is upregulated on cancer cells, resulting in cells that evade detection by the adaptive immune system. In particular, PD-L1 can be upregulated in MM in response to TLR agonists such as IFNγ and LPS. PD-L2 has much more selective expression compared to PD-L1. It is present in a subset of B cells and is upregulated on selected cells in response to strong NF-κB or STAT6 signaling.

  SMCs also function in T cells of SMC-treated mice both in vitro and in vivo, eg, increased proliferation of activated T cells extracted from mouse spleen after exposure to SMCs, increased cytokine production, And can affect higher cytokine production from NKT and NK cells. In addition, mice treated with SMCs show an excessive response to T cells upon antigen stimulation. Thus, SMC-based combination therapies can not only increase MM cell apoptosis but also stimulate selective adaptive responses. The combination of SMC and innate immune stimulators or immune checkpoint inhibitors (ICIs) may be the best means to overcome the strong survival promoting signals that MM cells accept.

  Cancer cells can manipulate many of the survival promotion strategies used to make healthy cells resistant to cell death induction signals. MM cells can specifically amplify constitutive NF-κB signaling used in plasma cells that make them resistant to apoptotic stimuli. This is achieved by increased expression of survival promoting NF--B target genes such as IL-6 and TNFα. Furthermore, MM cells can enhance the expression of checkpoint inhibitors, which are probably used to protect the cells from the inflammatory and cytotoxic environment; they cause detection by T cells and NK cells. Help to avoid. Targeting of both apoptotic resistance and immune evasion in MM has the ability to overcome two of the major aspects of treatment resistance in this disease.

  PD-1 blockade is significantly effective at delaying MM disease progression and improving mouse survival time, as shown using the syngeneic mouse MM model. The use of monoclonal antibodies to PD-1 has several advantages over alternatives to immune checkpoint blockade. First, it can block the binding of both PD-1 ligands, PD-L1 and PD-L2. Many cancers can upregulate PD-L1 in response to interferon treatment, and PD-1 / PD-L1 is upregulated in MM patients after treatment. Furthermore, a subset of immature B cells, called B1 cells, which secrete nonspecific antibodies showed high expression of PD-L2. Furthermore, PD-L2 expression can be increased in response to certain stimuli such as NF--B and STAT6 activation, demonstrating the importance of examining the expression levels of both ligands on MM cells. Human MM cells can upregulate both PD-1 ligands, making them unique as compared to solid tumors. This is a monoclonal antibody therapy targeting only PD-L1 (BMS-936559 / MDX-1105 from Bristol-Myers Squibb, MPDL 3280A from Genentech, MEDI 473 from MedImmune, and Averumab from EMD Serono) Although less effective than treatments targeting 1 (Bristol-Myers Squibb's nivolumab, Merck's penbrolizumab, and Curetech's pizirizumab), it demonstrates the value of using anti-PD-1 antibodies in MM.

  Second, the PD-1 targeting approach has the potential to have a more robust response to cancer compared to other ICIs such as anti-CTLA-4. The differences in activity may be attributed to the specific role of these molecules in T cell regulation. PD-1 is often found on CD8 + T cells and encountering its ligand inhibits the cytotoxic response activated by TCR signaling. In contrast, CTLA-4 has a more prominent role in secondary lymphoid tissues on regulatory T cells. The encounter of CTLA-4 with its receptor, CD28, beats or even downregulates the activating ligand for CD28 and blunts T cell secondary clonal expansion. The lack of efficacy of anti-CTLA-4 treatment in Example 3 is generally possible to indicate the invasion of MM into secondary lymphoid organs. This may impair the efficacy of anti-CTLA-4 by proportionally lower CD4 + T cell populations within germinal centers, or T cell infiltration into secondary lymphoid organs that are inhibited. In extramedullary MM, cells can form plasmacytomas in the spleen and lymph nodes often found in the late stages of the MM mouse model discussed in Example 3. Thus, it is clear that germinal centers are impaired by MPC-11 cells.

SMC
SMCs of the present invention can be any of the cIAP1, cIAP2 and / or XIAP, and optionally, one or more additional endogenous Smac activities that can be or can be expected to be inhibited. It may be a small molecule, a compound, a polypeptide, a protein, or any complex thereof. The SMCs of the invention can promote apoptosis by mimicking one or more activities of endogenous Smac, such as, but not limited to, the inhibition of cIAP1 and the inhibition of cIAP2. Endogenous Smac activity may be, for example, interaction with a particular protein, inhibition of a function of a particular protein, or inhibition of a particular IAP. In certain embodiments, SMCs inhibit both cIAP1 and cIAP2. In some embodiments, in addition to cIAP1 and cIAP2, SMC also inhibit one or more other IAPs such as XIAP or Livin / ML-IAP, an IAP containing a single BIR. In certain embodiments, SMCs inhibit cIAP1, cIAP2, and XIAP. In any of the embodiments comprising SMCs and an immunostimulatory agent, SMCs having specific activities can be selected for combination with one or more specific immunostimulatory agents. In any of the embodiments of the invention, SMCs can perform activities that Smac can not do. In some instances, these additional activities can contribute to the efficacy of the methods or compositions of the present invention.

  Treatment with SMC can deplete cIAP1 and cIAP2 cells, for example, by induction of self ubiquitination or trans ubiquitination and proteasome mediated degradation. SMCs can also suppress XIAP inhibition of caspases. SMCs function primarily by targeting cIAPs 1 and 2 and also by converting TNFα and, for example, for cancer cells, other cytokines or cell death ligands from survival signals to cell death signals be able to.

  Certain SMCs inhibit at least XIAP and cIAP. Such "pan-IAP" SMCs may mediate at several different yet interrelated stages of programmed cell death inhibition. This feature allows multiple cell death pathways to be affected by such SMCs and enables synergy with naturally occurring and emerging cancer therapeutics activating various apoptotic pathways that may be mediated by SMC. To minimize the opportunity for cancer to develop resistance to treatment with Pan-IAP SMC.

  One or more proinflammatory cytokines or cell death ligands, such as TNFα, TRAIL, and IL-1β, potently synergize with SMC therapy in many tumor-derived cell lines. In particular, strategies for increasing cell death ligand concentrations in SMC-treated tumors using techniques that limit toxicity commonly associated with recombinant cytokine therapy are thus very attractive. TNFα, TRAIL, and many other cytokines and chemokines can be upregulated in response to pathogen recognition by the subject's innate immune system. Importantly, this ancient response to microbial pathogens is usually self-limiting and safe for the subject, due to the strict negative regulation that limits the intensity and duration of its activity.

  SMC can be rationally designed based on Smac. The ability of a compound to promote apoptosis by mimicking one or more functions or activities of endogenous Smac can be predicted based on similarity to endogenous Smac or known SMCs. The SMC may be a compound, a polypeptide, a protein, or a complex of two or more compounds, polypeptides, or proteins.

  In some instances, SMCs are small molecule IAP antagonists based on the N-terminal tetrapeptide sequence of the Smac polypeptide (shown after processing). In some instances, the SMCs are monomeric (monovalent) or dimeric (divalent). In particular examples, SMCs are AVPIs derived from Smac / DIABLO, the second mitochondrial activator of caspases, or other similar IBM (eg, IAP binding motifs from other proteins such as casp9) And one or two moieties that mimic the tetrapeptide sequence of The SMC dimers of the present invention may be homodimers or heterodimers. In certain embodiments, the dimeric subunits are anchored by various linkers. The linker may be in the same defined spot of any subunit but may be at a different fixation point ("aa" position P1, P2, P3 or P4 and sometimes P5 group is also available ) May be located. In various configurations, the dimer subunits may be in different orientations, for example head to tail, head to head, or tail to tail. The heterodimer may comprise two different monomers with different BIR domains or different affinity for different IAPs. Alternatively, the heterodimer may comprise Smac monomer and a ligand for another receptor or target that is not IAP. In some instances, the SMC may be cyclic. In some instances, SMCs may be trimers or multimers. The multimerized SMC is 10, 20, 30, 40, 50, 100, 200, 1,000, 5,000, 7,000 or more (e.g., by EC50 in vitro) compared to one or more corresponding monomers It may exhibit a 7,000-fold or more increase in activity, etc.). In some instances this may occur, for example, because tethering enhances ubiquitination between IAPs, or because dual BIR binding enhances the stability of the interaction. While multimers such as dimers may exhibit increased activity, in some embodiments monomers are preferred. For example, in some instances, low molecular weight SMCs are preferred, for example, for reasons associated with bioavailability.

  In some instances of the invention, the agent capable of inhibiting cIAP1 / 2 is bestatin or a Me-bestatin analog. The bestatin or Me-bestatin analog can induce cIAP1 / 2 autoubiquitination and mimic the biological activity of Smac.

  In certain embodiments of the invention, the SMC combination treatment comprises one or more SMCs and one or more interferon agents, such as a type 1 interferon agent, a type 2 interferon agent, and a type 3 interferon agent. Including. Combination treatments comprising an interferon agent may be useful in the treatment of cancer such as multiple myeloma.

  In some embodiments, a VSV expressing a gene that enables imaging, such as NIS that expresses IFN, optionally the sodium-iodine cotransporter, is used in combination with SMC. For example, such VSV can be used in combination with an SMC, such as the Ascentage Smac mimic SM-1387 / APG-1387, the Novartis Smac mimic LCL 161, or the birinapant. Such combinations may be useful in the treatment of cancer, such as hepatocellular carcinoma or liver metastasis.

  Various SMCs are known in the art. Non-limiting examples of SMC are provided in Table 1 (Table 1). Table 1 includes proposed mechanisms by which various SMCs can function, but the methods and compositions of the present invention are not limited by or by these mechanisms.

Agents The immunostimulatory agent or immunomodulator of the invention may be any agent capable of inducing a receptor-mediated apoptotic program that is inhibited by cIAP1 and cIAP2 in one or more cells of a subject . The immunostimulatory agents of the invention may be cIAP1 (BIRC2), cIAP2 (BIRC3 or API2), and optionally one or more additional IAPs, such as one or more human IAP proteins NAIP (BIRC1), XIAP (XIAP). An apoptotic program may be induced that is regulated by BIRC4), survivin (BIRC5), apollon / blues (BIRC6), ML-IAP (BIRC7 or livin), and ILP-2 (BIRC8). It is further known that various immunomodulators, such as CpG or IAP antagonists, can alter the status of immune cells.

  In some instances, the immunostimulatory agent may be a TLR agonist, such as a TLR ligand. The TLR agonists of the present invention can be used in TLR-1, TLR-2, TLR-3, TLR-4, TLR-5, TLR-6, TLR-7, TLR-8, TLR-9, and TLR-10 in humans. It may be an agonist of one or more of the related proteins, or related proteins (eg, mouse TLR-1 to TLR-9 and TLR-11 to TLR-13) in other species. TLRs are highly conserved known as pathogen-associated microbial patterns (PAMPs) expressed exclusively by microbial pathogens, and hazard-associated molecular patterns (DAMPs), which are endogenous molecules released from necrotic cells or dying cells. It can recognize structural motifs. PAMP includes various bacterial cell wall components such as lipopolysaccharide (LPS), peptidoglycan (PGN), and lipopeptides, as well as flagellin, bacterial DNA, and viral duplex RNA. DAMP includes intracellular proteins such as heat shock proteins as well as protein fragments derived from the extracellular matrix. The agonist of the present invention further comprises, for example, CpG oligodeoxynucleotides (CpG ODN) such as class A, B and C CpG ODN, nucleic acids such as base analogs, dsRNA or pathogen DNA, or pathogens or pathogen-like cells or virions Including. In certain embodiments, the agent is an agent that mimics a virus or bacteria, or is a synthetic TLR agonist.

  Various TLR agonists are known in the art. Nonlimiting examples of TLR agonists are provided in Table 2. Table 2 includes proposed mechanisms, uses, or TLR targets that various TLR agonists may function, but the methods and compositions of the present invention depend on these mechanisms, uses, or targets. Or it is not limited to them.

  In another example, the immunostimulatory agent may be a virus, eg, an oncolytic virus. Oncolytic viruses are viruses that selectively infect, replicate, and / or kill cancer cells selectively. The viruses of the present invention include, but are not limited to, adenovirus, herpes simplex virus, measles virus, Newcastle disease virus, parvovirus, polio virus, reovirus, Seneca valley virus, retrovirus, vaccinia virus, varicella Stomatitis virus, lentivirus, rhabdovirus, sindbis virus, coxsackie virus, pox virus and others can be mentioned. In certain embodiments of the invention, the agent is a rhabdovirus, eg, VSV. Rhabdovirus can replicate rapidly with high IFN production. In another particular embodiment, the agent is a wild virus, such as Maraba virus, Farmington virus, Carajas virus, having a MG1 double mutation. The viral agent of the present invention may be a virus mutant (eg, Matrix or VSV having Δ51 mutation in M protein), a transgene modified virus (eg, VSV-hIFNβ), TNFα, LTα / TNFβ, TRAIL, FasL, Included are viruses bearing TL1α, chimeric viruses (eg, rabies), or pseudotyped viruses (eg, viruses pseudotyped with G proteins derived from LCMV or other viruses). In some instances, viruses of the invention are selected to reduce neurotoxicity. Common viruses, in particular oncolytic viruses, are known in the art.

  In certain embodiments, the agent is a killed VSV NRRP particle or a prime boost tumor vaccine. NRRPs are wild-type VSV modified to produce infectious vectors that no longer replicate or spread but retain oncolytic and immunostimulatory properties. NRRP can be produced using gamma irradiation, UV or busulfan. Specific combination therapies include prime boost with Adeno-MAGE3 (melanoma antigen) and / or Maraba-MG1-MAGE3. Other particular combination therapies include wild type VSV NRRP killed by UV or killed by gamma irradiation. NRRP exhibits low or no neurotoxicity. NRRPs may be useful, for example, in the treatment of gliomas, hematologic (liquid) tumors, or multiple myelomas.

  In some cases, the agent of the invention is a vaccine strain, an attenuated virus or microorganism, or a killed virus or microorganism. In some cases, the agent may be, for example, BCG, a live or dead rabbi vaccine, or an influenza vaccine.

  Non-limiting examples of viruses of the invention, eg, oncolytic viruses, are provided in Table 3. Table 3 includes the proposed mechanisms or uses of the provided viruses, but the methods and compositions of the present invention are not limited by or to these mechanisms or uses.

Cancer The methods and compositions of the present invention can be used to treat various cancer types. The methods and compositions of the present invention have broad applicability to many if not all cancers since those skilled in the art can perform receptor-mediated apoptosis in many if not all cells. I understand that it is possible. The combination approach of the present invention is effective in various aggressive treatment refractory tumor models. In certain embodiments, for example, the cancer treated by the methods of the present invention may be adrenal cancer, basal cell cancer, biliary cancer, bladder cancer, osteosarcoma, brain tumors and other central nervous system (CNS) Cancer, Breast cancer, Cervical cancer, Choriocarcinoma, Colon cancer, Colorectal cancer, Connective tissue cancer, Gastrointestinal cancer, Endometrial cancer, Nasopharyngeal cancer, Esophageal cancer, Eye Lymphoma including cancer, gallbladder cancer, stomach cancer, head and neck cancer, hepatocellular carcinoma, intraepithelial neoplasia, kidney cancer, laryngeal cancer, leukemia, liver cancer, liver metastasis, lung cancer, Hodgkin's and non-Hodgkin's lymphoma , Melanoma, myeloma, multiple myeloma, neuroblastoma, mesothelioma, glioma, myelodysplastic syndrome, multiple myeloma, oral cancer (eg, lip, tongue, mouth, and pharynx), Ovarian cancer, childhood cancer, pancreatic cancer, pancreatic endocrine tumor, penile cancer, plasma cell tumor, pituitary adenoma, prostate cancer, renal cell Cancer, respiratory system cancer, rhabdomyosarcoma, salivary gland cancer, sarcoma, skin cancer, small intestine cancer, stomach cancer, testicular cancer, thyroid cancer, ureteral cancer, urological cancer, It may be other carcinomas and sarcomas. Other cancers are known in the art.

  The cancer may be a cancer that is refractory to treatment with SMC alone. The methods and compositions of the present invention may be particularly useful in cancers that are refractory to treatment with SMC alone. Typically, cancers that are refractory to treatment with SMC alone may be cancers in which the IAP-mediated apoptotic pathway is not significantly induced. In certain embodiments, the cancer of the present invention is in such a manner that one or more apoptotic pathways are not significantly induced, ie, treatment with SMC alone is sufficient to effectively treat the cancer. It is a cancer that is not activated. For example, the cancer of the present invention may be a cancer in which the cIAP1 / 2-mediated apoptotic pathway is not significantly induced.

  The cancer of the present invention may be refractory to treatment with one or more agents. In certain embodiments, the cancer of the invention is refractory to treatment with one or more agents (without SMC) and is also for treatment with one or more SMCs (without agent). It may be a cancer that is refractory.

Formulation and Administration In some cases, delivery of naked and natural forms of SMCs and / or agents may be sufficient to promote apoptosis and / or treat cancer. The SMC and / or the drug, provided that the salt, ester, amide, prodrug or derivative is suitably pharmacologically effective, eg promoting apoptosis and / or treating cancer. Can be administered in the form of salts, esters, amides, prodrugs, derivatives and the like.

  Salts, esters, amides, prodrugs and other derivatives of SMCs or drugs can be prepared using standard procedures known in the art of synthetic organic chemistry. For example, acid salts of SMCs and / or agents can be prepared from SMCs or agents in free base form, typically using conventional methods involving reaction with a suitable acid. Generally, the SMC in basic form or the drug is dissolved in a polar organic solvent such as methanol or ethanol and the acid is added thereto. The resulting salt may precipitate or may be removed from solution by the addition of a less polar solvent. Suitable acids for preparing the acid addition salt include, but are not limited to, organic acids such as acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, malic acid, malonic acid, succinic acid, Maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid etc., as well as inorganic acids such as hydrochloric acid, hydrobromic acid, Both sulfuric acid, nitric acid, phosphoric acid and the like can be mentioned.

  The acid addition salts can be converted to the free base by treatment with a suitable base. Certain typical acid addition salts of SMC and / or agents, such as, for example, halide salts, can be prepared using hydrochloric acid or hydrobromic acid. Conversely, the preparation of the basic salt of SMC and / or drug of the present invention is carried out similarly using a pharmaceutically acceptable base such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, trimethylamine etc. It can be prepared in the manner of Certain specific base salts include, but are not limited to, alkali metal salts such as sodium salts and copper salts.

  Preparation of the ester may include, for example, functionalization of hydroxyl and / or carboxyl groups present in the molecular structure of SMC and / or the drug. In certain embodiments, the ester is a free alcohol group, ie, an acyl-substituted derivative of a moiety derived from a carboxylic acid of the formula RCOOH, where R is alkyl, preferably lower alkyl. If desired, the ester can be reconverted to the free acid by using conventional hydrogenolysis or hydrolysis procedures.

  The amides can also be prepared using techniques known in the art. For example, the amide can be prepared from the ester using a suitable amine reactant, or from the anhydride or acid chloride by reaction with ammonia or lower alkylamine.

  The SMCs or agents of the invention can be combined with a pharmaceutically acceptable carrier (excipient) to form a pharmaceutical composition. The pharmaceutically acceptable carrier acts, for example, to stabilize the composition, to increase or decrease the absorption of SMC or drug, or to improve the penetration of the blood-brain barrier (if necessary). , One or more physiologically acceptable compounds. Physiologically acceptable compounds are, for example, carbohydrates (for example glucose, sucrose or dextran), antioxidants (for example ascorbic acid or glutathione), chelating agents, low molecular weight proteins, protection and uptake enhancers (for example Lipids), compositions that reduce the clearance or hydrolysis of the active agent, or excipients or other stabilizers and / or buffers. In particular, other physiologically acceptable compounds useful in the preparation of tablets, capsules, gelcaps and the like include, but are not limited to, binders, diluents / fillers, disintegrants, lubricants, suspensions, etc. Turbid agents etc. may be mentioned. In certain embodiments, a pharmaceutical formulation can enhance the delivery or efficacy of SMC or a drug.

  In various embodiments, the SMCs or agents of the invention can be prepared for parenteral, topical, oral, nasal (or otherwise inhaled), rectal, or topical administration. Administration can be performed, for example, transdermally, prophylactically or by aerosol.

  The pharmaceutical composition of the present invention can be administered in various unit dosage forms depending on the method of administration. Suitable unit dosage forms include, but are not limited to, powders, tablets, pills, capsules, lozenges, suppositories, patches, nasal sprays, injectables, implantable sustained release formulations, and lipid complexes Can be mentioned.

  In certain embodiments, excipients (eg, lactose, sucrose, starch, mannitol, etc.), optionally disintegrants (eg, calcium carbonate, carboxymethylcellulose calcium, sodium starch glycolate, crospovidone etc.), binding agents (eg, For example, alpha starch, gum arabic, microcrystalline cellulose, carboxymethylcellulose, polyvinyl pyrrolidone, hydroxypropyl cellulose, cyclodextrin etc., or optionally a lubricant (eg talc, magnesium stearate, polyethylene glycol 6000 etc.), SMC Alternatively, it can be added to a drug and the resulting composition can be compressed to produce an oral dosage form (eg, a tablet). In certain embodiments, the compressed product is coated to, for example, mask the taste of the compressed product, promote intestinal dissolution of the compressed product, or provide sustained release of SMC or drug Can be promoted. Suitable coating materials include, but are not limited to, ethylcellulose, hydroxymethylcellulose, polyoxyethylene glycol, cellulose acetate phthalate, hydroxypropyl methylcellulose phthalate, and Eudragit (Rohm & Haas, Germany; methacrylic acid-acrylic) Acid copolymers).

  Other physiologically acceptable compounds that can be included in SMC or pharmaceutical compositions containing the drug are wetting agents, emulsifiers, dispersants or preservatives that are particularly useful for preventing the growth or activity of microorganisms May be included. Various preservatives are well known and include, for example, phenol and ascorbic acid. The choice of a pharmaceutically acceptable carrier, including a physiologically acceptable compound, depends, for example, on the administration route of the SMC or agent and the particular physicochemical characteristics of the SMC or agent.

  In certain embodiments, one or more excipients for use in a pharmaceutical composition comprising SMC or a medicament is one that is substantially free of sterile and / or undesired substances. You may Such compositions can be sterilized by conventional techniques known in the art. No sterilization is necessary for various oral dosage forms excipients, such as tablets and capsules. Standards, eg, USP / NF standards, are known in the art.

  Single or multiple doses of the SMC or pharmaceutical composition of the invention, depending on the dose, the required frequency of administration, and the subject's known or expected tolerance of the composition regarding the dose and frequency of administration. It can be administered by single administration. In various embodiments, the composition can provide a sufficient amount of the SMCs or agents of the present invention to effectively treat cancer.

  The amount and / or concentration of SMCs or agents administered to a subject may vary widely, typically the activity of the SMCs or agents and the characteristics of the subject, eg, species and body weight, and the particular mode of administration and For example, they are selected based primarily on the subject's need for the type of cancer. The dose can be varied to optimize the therapeutic and / or prophylactic regimen in a particular subject or group of subjects.

  In certain embodiments, the SMCs or agents of the invention are administered to the oral cavity by use of, for example, lozenges, aerosol sprays, mouth washes, coated swabs, or other mechanisms known in the art.

  In certain embodiments, the SMCs or agents of the invention are administered using a slow release solid wafer that is inserted into a cavity in the brain during tumor resection at the time of surgery. The wafer may be a biodegradable polyanhydride wafer containing SMC or poly (I: C). The number of wafers placed may be dependent on the size of the post-surgical excision of the primary brain tumor. Delivery of drugs directly from sustained release wafers to brain tissue avoids the problem of delivering systemic treatments that cross the blood-brain barrier. The polymer matrix is dissolved with the drug in an organic solvent, spray-dried to fine particles in the range of 1 to 20 μm, and compression molded to a wafer, 1,3-bis- (p-carboxyphenoxy) propane and sebacic acid It may be composed of a copolymer (PCPP-SA; 80:20 molar ratio). In one particular embodiment, the rigid wafer degrades in a two-step process in which the copolymer erodes into the surrounding aqueous environment after water penetration has hydrolyzed the anhydride bonds during the first 10 hours.

  In certain embodiments, SMCs or agents of the invention can be administered systemically (eg, orally or as an injection) according to standard methods known in the art. In certain embodiments, using a transdermal drug delivery system, ie, a transdermal "patch", wherein the SMC or drug is contained in a layered structure that serves as a drug delivery device that is typically fixed to the skin. Thus, SMC or a drug can be delivered through the skin. In such structures, the drug composition is typically contained in the lower layer or reservoir of the upper back layer. The reservoir of the transdermal patch contains the amount of SMC or drug that is ultimately available for delivery to the surface of the skin. Thus, the reservoir may comprise, for example, the SMCs or agents of the invention in an adhesive on the back layer of the patch or in any of a variety of different matrix formulations known in the art. The patch may contain a single reservoir or multiple reservoirs.

  In certain transdermal patch embodiments, the reservoir may comprise a polymer matrix of a pharmaceutically acceptable contact adhesive material which serves to secure the system to the skin during drug delivery. Examples of suitable skin contact adhesive materials include, but are not limited to, polyethylene, polysiloxanes, polyisobutylenes, polyacrylates, and polyurethanes. Alternatively, the reservoir containing SMC and / or the drug and the skin-contacting adhesive are present as separate and distinct layers, the adhesive at the bottom of the reservoir being, in this case, a polymer matrix, liquid or hydrogel reservoir as described above, Or it may be another form of reservoir known in the art. The backing layer in these laminates, which serves as the upper surface of the device, preferably functions as the primary structural element of the patch, providing the device with a substantially flexible portion. The material selected for the backing layer is preferably substantially impermeable to the SMC and / or the drug and to any other material present.

  Additional formulations for topical delivery include, but are not limited to, ointments, gels, sprays, fluids, and creams. Ointments are semisolid preparations, typically based on petrolatum or other petroleum derivatives. Creams containing SMCs or agents are typically viscous liquid or semisolid emulsions, such as oil-in-water or water-in-oil emulsions. Cream bases are typically water-washable and comprise an oil phase, an emulsifier and an aqueous phase. The oil phase of a cream base, also referred to as the "inner" phase, is generally composed of petrolatum and a fatty alcohol such as cetyl alcohol or stearyl alcohol; the aqueous phase is usually, but not necessarily, It exceeds the oil phase in volume and generally contains a humectant. The emulsifier in a cream formulation is generally a nonionic, anionic, cationic or amphoteric surfactant. The particular ointment or cream base used can be selected to provide optimal drug delivery by those skilled in the art. For other carriers or vehicles, an ointment base may be inert, stable, nonirritating, and nonsensitizing.

  Various buccal and sublingual formulations are also contemplated.

  In certain embodiments, administration of SMCs or agents of the invention may be parenteral. Parenteral administration may include intrathecal, epidural, intrathecal, subcutaneous or intravenous administration. Means of parenteral administration are known in the art. In certain embodiments, parenteral administration may include a device implanted subcutaneously.

  In certain embodiments, it is desirable to deliver SMCs or agents to the brain. In embodiments involving systemic administration, this requires that the SMC or drug cross the blood-brain barrier. In various embodiments, this is by co-administering the SMC or drug with a carrier molecule, such as a cationic dendrimer or arginine rich peptide, capable of delivering the SMC or drug across the blood brain barrier. It can be easy.

  In certain embodiments, the SMC or agent is implanted or partially implanted by administration via implantation of a biocompatible release system (eg, a reservoir), direct administration via an implanted cannula, or It can be delivered directly to the brain by administration via a drug pump or by similar functional mechanisms known in the art. In certain embodiments, SMCs or agents can be administered systemically (eg, injected intravenously). In certain embodiments, transporting SMCs or drugs across the blood brain barrier without using additional compounds included in the pharmaceutical composition to enhance transport across the blood brain barrier. There is expected.

  In certain embodiments, one or more SMCs or agents of the invention are ready for dilution or addition thereto, eg, with a volume of water, alcohol, hydrogen peroxide, or other diluents It can be provided as a concentrate in storage containers or soluble capsules. The concentrate of the present invention can be provided in a specific amount of SMC or drug and / or a specific total amount. The concentrate can be formulated for dilution in a specific volume of diluent prior to administration.

  The SMC or agent can be administered orally in the form of tablets, capsules, elixirs or syrups, or rectally in the form of suppositories. The compounds can also be administered topically in the form of foams, lotions, drops, creams, ointments, emollients, or gels. Parenteral administration of the compounds is suitably carried out, for example, in the form of a saline solution or with the compound incorporated into liposomes. If the compound itself is not sufficiently soluble to dissolve, solubilizers such as ethanol can be applied. Other suitable formulations and modes of administration are known in the art or may be derived from the art.

  The SMCs or agents of the invention can be administered to a mammal in need thereof, such as a mammal diagnosed as having cancer. The SMCs or agents of the invention can be administered to promote apoptosis and / or treat cancer.

  The therapeutically effective dose of the pharmaceutical composition of the invention may depend on the age of the subject, the sex of the subject, the species of the subject, the particular pathology, the severity of the symptoms and the general state of health of the subject.

  The present invention includes compositions and methods for the treatment of human subjects, such as human subjects diagnosed with cancer. Furthermore, the pharmaceutical composition of the invention may be suitable for administration to animals, for example for veterinary use. Certain embodiments of the present invention are directed to medicaments of the invention for non-human organisms, such as non-human primates, dogs, horses, cats, pigs, ungulates, or animals of the order or other vertebrate species. Administration of the composition may be included.

  The therapy according to the invention can be performed alone or in combination with another therapy, for example another cancer therapy, and can be provided at home, a doctor's office, a clinic, an outpatient department of a hospital, or a hospital. In some cases, treatment may be initiated at the hospital or on an outpatient basis so that the physician can closely observe the effects of the therapy and make the necessary adjustments. The duration of therapy depends on the type of disease or disorder being treated, the age and condition of the subject, the stage and type of disease of the subject, and how the patient responds to the treatment.

  In certain embodiments, the combination of therapies of the invention further comprises treatment with a recombinant interferon, such as IFN-α, IFN-β, IFN-γ, pegylated IFN, or liposomal interferon. In some embodiments, the combination of therapies of the invention further comprises treatment with recombinant TNF-α, for example, for limb perfusion. In certain embodiments, the combination therapies of the invention further comprise treatment with one or more TNF-α or IFN inducing compounds, such as DMXAA, ribavirin and the like. Additional cancer immunotherapies that can be used in combination with the present invention include antibodies that target CTLA-4, PD-1, PD-L1, PD-L2, or other checkpoint inhibitors, eg, monoclonal antibodies Including antibodies. Cyclic dinucleotide (CDN) [cyclic di-GMP (guanosine 5'-monophosphate) (CDG), cyclic diAMP (adenosine 5'-monophosphate) (CDA), and cyclic GMP-AMP (cGAMP)] , A class of pathogen-related molecular pattern molecules (PAMPs) that activate TBK1 / interferon regulatory factor 3 (IRF3) / type 1 interferon (IFN) signaling axis by the cytoplasmic pattern recognition receptor stimulating factor (STING) of the interferon gene . In certain embodiments, STING agonists can be combined with SMCs to treat cancer.

  Routes of administration for the various embodiments include, but are not limited to, topical, transdermal, nasal, and systemic administration (intravenous, intramuscular, subcutaneous, inhalation, rectal, buccal, vaginal, intraperitoneal Intra-articular, intra-articular, ocular, aural or oral administration). As used herein, "systemic administration" refers to all non-dermal routes of administration, and specifically does not include topical and transdermal routes of administration.

  In any of the above embodiments, the route of administration can be optimized based on the characteristics of the SMC or drug. In some instances, the SMC or agent is a small molecule or compound. In another example, the SMC or agent is a nucleic acid. In yet another example, the agent may be a cell or a virus. In any of these or other embodiments, the appropriate formulation and route of administration will be selected in accordance with the art.

  In an embodiment of the invention, SMCs and agents are administered to a subject in need thereof, eg, a subject with cancer. In some instances, the SMC and the agent are administered simultaneously. In some embodiments, the SMC and the agent may be present in a single therapeutic dosage form. In another embodiment, the SMC and the agent may be administered separately to a subject in need thereof. When administered separately, the SMC and agent may be administered simultaneously or at different times. In some instances, the subject receives a single dose of SMC and a single dose of drug. In certain embodiments, one or more SMCs and an agent are administered to the subject at two or more doses. In certain embodiments, the frequency of administration of SMC and the frequency of administration of drug are non-identical, ie, SMC is administered at a first frequency and the drug is administered at a second frequency.

  In some embodiments, SMC is administered within one week of administration of the agent. In certain embodiments, SMCs are administered within 3 days (72 hours) of administration of the agent. In an even more specific embodiment, SMC is administered within one day (24 hours) of administration of the agent.

  In certain embodiments of any of the methods of the invention, the SMC and agent are administered within 28 days of each other, or less, for example, within 14 days of each other. In certain embodiments of any of the methods of the invention, the SMC and agent are, for example, simultaneously, or one minute, five minutes, ten minutes, fifteen minutes, thirty minutes, one hour, two hours, four hours, or each other. It is administered within 6 hours, 12 hours, 18 hours, 24 hours, 36 hours, 2 days, 4 days, 8 days, 10 days, 12 days, 16 days, 20 days, 24 days or 28 days. In any of these embodiments, the first administration of the SMC of the invention may be prior to the first administration of the agent of the invention. Alternatively, in any of these embodiments, the first administration of the SMC of the invention may be after the first administration of the agent of the invention. Also, as the SMCs and / or agents of the invention may be administered to a subject in more than one dose, and in such instances, the doses of SMCs and agents of the invention may be administered at different frequencies. For good, it is not necessary that the time between the administration of SMC and the administration of the drug remain constant within a given treatment course or for a given subject.

  One or both of the SMC and the drug can be administered at low or high doses. In the embodiment in which the SMC and the drug are separately formulated, the pharmacokinetic profile of each drug can be suitably matched with the formulation, dose, administration route and the like. In some instances, SMC is administered at a standard or high dose and the drug is administered at low doses. In some instances, SMCs are administered at low doses and agents are administered at standard or high doses. In some cases, both the SMC and the drug are administered at standard doses or at high doses. In some instances, both the SMC and the drug are administered at low doses.

  The dose and frequency of administration of each component of the combination can be controlled independently. For example, one component may be administered three times a day, while the second component may be administered once a day, or one component may be administered once a week, but the second component may be administered once every two weeks. It can be administered. The combination therapy may be administered on and off cycles, including rest periods, so that the subject's body has a chance to recover from the effects of treatment.

Kits In general, the kits of the invention contain one or more SMCs and one or more agents. These may be provided in the kit as separate compositions or may be combined in a single composition as described above. Kits of the invention may also contain instructions for the administration of one or more SMCs and one or more agents.

  Kits of the invention also contain instructions for administering additional pharmacologically acceptable substances, such as agents known to treat cancer that are not SMCs or agents of the invention. It is also good.

  The separately or separately formulated agents can be packaged together as a kit. Nonlimiting examples include kits containing, for example, two pills, pills and powders, liquids in suppositories and vials, two topical creams, ointments, foams, and the like. The kit may also contain any component that aids in the administration of the unit dose to the subject, such as a vial to reconstitute the powder form, a syringe for injection, a customized IV delivery system, an inhaler etc. Good. In addition, unit dose kits may contain instructions for preparation and administration of the composition. The kit may be used as a single-use unit dose for a subject, as multiple uses for a particular subject (in a fixed dose regimen, or as individual compounds may vary in efficacy as therapy progresses) The kit can be manufactured; or the kit may contain multiple doses suitable for administration to multiple subjects ("bulk package"). The components of the kit can be assembled into cartons, blister packs, bottles, tubes and the like.

  The dose of each compound in the claimed combination depends on the method of administration, the disease being treated (eg, type of cancer), the severity of the disease, and the age, weight, and health of the individual being treated, etc. It depends on the factor. Furthermore, the pharmacogenomic (the effect of the genotype on the pharmacokinetics, pharmacodynamics or efficacy profile of the therapeutic agent) information for a particular subject may influence the dosage regimen or other aspects of administration.

Example 1
Smac mimics prime tumors for destruction by the innate immune system
Smac mimetic compounds are a class of apoptosis sensitizers that have been shown to be safe in Phase I clinical trials of cancer patients. Stimulation of the innate anti-pathogen response can generate a powerful but safe inflammatory "cytokine storm" which induces the death of the tumor treated with the Smac mimic. This example demonstrates treatment with Smac mimetics in a manner in which activation of the innate immune response by an oncolytic virus and an adjuvant such as poly (I: C) and CpG is mediated by IFNβ, TNFα or TRAIL. To demonstrate that they induce bystander cell death in cancer cells. This therapeutic strategy can provide permanent cure, for example, in some aggressive mouse models of cancer. With regard to these and other innate immune stimulators that have shown safety in human clinical trials, the data provided herein strongly points to their combined use with Smac mimetics to treat cancer.

  In this example, stimulation of the innate immune system using pathogen mimics is a safe and effective strategy to generate the cytokine environment necessary to initiate apoptosis in tumors treated with SMC It is to check if it is. We here rely on either non-pathogenic oncolytic virus, and mimics of microbial RNA or DNA, such as poly (I: C) and CpG, to produce either IFN beta, TNF alpha or TRAIL We report to induce bystander killing of cancer cells treated with SMC. Importantly, this therapeutic strategy has been tolerated in vivo and has resulted in a permanent cure in several aggressive mouse models of cancer.

SMC therapy sensitizes cancer cells to bystander cell death during oncolytic virus infection Oncolytic virus (OV) is a new organism for cancer currently in Phase I-III clinical evaluation. It is a therapy. One barrier to OV therapy may be the induction of type I IFN and NFκB responsive cytokines by the host that directs the antiviral status in tumors. We examined whether we could utilize these innate immune cytokines to induce apoptosis in cancer cells pretreated with SMC. First, a small panel (n = 30) of tumor-derived and normal cell lines was screened for responsiveness to SMC LCL 161 and the oncolytic rhabdovirus VSVΔ51. We select LCL 161 as this compound is the most clinically advanced drug in the SMC class and selects VSV Δ51 as it is known to induce a robust antiviral cytokine response. In 15 of the 28 cancer cell lines tested (54%), SMC treatment enhanced the sensitivity EC50 of VSV Δ51 10-10,000 fold (FIG. 6, and representative examples in FIGS. 1A and 1B). Similarly, low doses of VSVΔ51 have an EC50 of SMC therapy from indeterminate levels (> 2500 nM) to 4.5 and 21.9 nM in two representative cell lines: mouse mammary adenocarcinoma EMT6 and human glioblastoma SNB75 cells, respectively Decreased (Figure 1C). Combination factor analysis determined that the interaction between SMC therapy and VSV Δ51 was synergistic (Figure 7). Experiments using four other SMCs and five other oncolytic viruses showed that various monovalent and divalent SMCs synergize with VSVΔ51 (FIG. 8). We compared the HSV, reovirus, vaccinia and wild-type VSV platforms where the oncolytic rhabdovirus VSV Δ51 and Maraba-MG1 all altered the mechanism of disarming the mode of innate immune signaling. , It was found to be excellent in synergy with SMC to cause bystander killing (FIGS. 9A and 9B). Genetic experiments using RNAi-mediated silencing indicated that it is necessary to inhibit both XIAP and cIAP to obtain synergy with VSVΔ51 (FIG. 10A, FIG. 10B and FIG. 24C) . In sharp contrast to the results in tumor-derived cell lines, non-cancerous GM38 primary human dermal fibroblasts and HSkM human skeletal myoblasts were not affected by the combination therapy of VSVΔ51 and SMC (FIG. 6). Taken together, these data indicate that oncolytic VSV synergizes with SMC therapy in a tumor selective manner.

  To determine whether VSV Δ51 elicits bystander cell death in IAP-depleted neighboring cells not infected by virus, cells prior to infection with low dose VSV Δ51 (MOI = 0.01 infectious particles / cell) Was treated with SMC. We found cell death when conditioned medium from cells infected with VSVΔ51 (which is then inactivated by UV light) was transferred to a plate of SMC-treated virus naive cancer cells. It was evaluated whether it could be induced. Conditioned media induced cell death only when the cells were co-treated with SMC (FIG. 1D). We also used a low dose of VSV Δ51 pseudotyped non G-free strain (MOI = 0.1) containing a deletion of the gene encoding its glycoprotein (VSV Δ51 ΔG) which limits the virus to one round of infection. , Were also found to be toxic to all plates of SMC-treated cancer cells (FIG. 1E). Finally, we performed a cytotoxicity assay in agarose-covered cells used to delay the diffusion of VSV Δ51 expressing a fluorescent tag and treated with SMC outside the area of viral infection. Dramatic cell death was observed in these cells (FIG. 1F and FIG. 11). Overall, these results show that VSV Δ51 infection results in the release of at least one soluble factor that can potently induce bystander cell death in SMC-treated nearby, uninfected cancer cells. It shows.

SMC therapy does not impair cellular innate immune response to oncolytic VSV Cellular innate immune response to RNA virus infection in mammalian tumor cells, cytosolic (RIG-like receptor, RLR) and endosomes (Toll-like receptor, TLR) can be initiated by members of the viral RNA sensor family. Once triggered, these receptors can seed simultaneous IFN response factor (IRF) 3/7 and nuclear factor kappa B (NF-κB) cell signaling cascades. These signals can lead to the production of an array of IFN and its responsive genes as well as inflammatory chemokines and cytokines. This stimulates neighboring cells to preemptively express antiviral genetic warfare and also aids in mobilization and activation of cells within the innate and adaptive immune system, ultimately eliminating viral infection. cIAP proteins have recently been implicated in several signaling pathways downstream of pathogen recognition, including those originating from RLRs and TLRs. Therefore, it was examined whether SMC therapy alters the antiviral response to oncolytic VSV infection in tumor cells and mice. First, the effect of SMC therapy on the productivity and spread of VSV Δ51 was evaluated. Single-step and multi-step growth curves of VSVΔ51 productivity showed that SMC treatment does not affect the growth kinetics of VSVΔ51 in EMT6 or SNB75 cells in vitro (FIG. 2A). Furthermore, analysis by time-lapse microscopy shows that SMC treatment does not alter the infectivity or spread by tumor cells of VSV Δ51 in tumor cells (FIG. 2B). In addition, virus replication and spread in vivo was analyzed by determining tumor burden using IVIS imaging and tissue virus titration. No differences in virus kinetics were found upon SMC treatment in EMT6 tumor-bearing mice (FIGS. 12A and 12B). Because EMT6 cells and SNB75 cells both have functional type I IFN responses that modulate the VSV life cycle, these data indirectly indicate that SMC therapy does not affect the antiviral signaling cascade in cancer cells. But provide strong evidence.

  For further scrutiny, IFNβ production was measured in EMT6 and SNB75 cells treated with VSVΔ51 and SMC. This experiment showed that SMC-treated cancer cells respond to VSVΔ51 by secreting IFNβ (FIG. 2C), although at slightly lower levels compared to VSVΔ51 alone. It was investigated whether suppressed IFNβ secretion from SMC-treated cells was associated with the induction of downstream IFN-stimulated genes (ISG). Quantitative RT-PCR analysis of a small panel of ISG in cells treated with VSVΔ51 and SMC showed that IAP inhibition is not associated with ISG gene expression in response to oncolytic VSV infection (FIG. 2D) . Consistent with this finding, Western blot analysis showed that SMC did not alter activation of Jak / Stat signaling downstream of IFNβ (FIG. 2E and FIG. 24A). Collectively, these data suggest that SMCs sense infection from VSVΔ51 and do not inhibit the ability of tumor cells to respond to it.

IFN.BETA. Directs bystander cell death during concurrent treatment with SMC and oncolytic VSV
SMCs sensitize several cancer cell lines to caspase 8 dependent apoptosis induced by TNFα, TRAIL, and IL-1β. Because RNA viruses can induce the production of these cytokines as part of a cellular antiviral response, we investigated the involvement of cytokine signaling in cell death induced by SMC and OV. First, TNF receptor (TNF-R1) and / or TRAIL receptor (DR5) were silenced to assay the synergy between SMC and VSVΔ51. This experiment showed that not only TNFα and TRAIL are involved but, collectively, they are essential for bystander cell death (FIGS. 3A-3H, 13A and 24D). Consistent with this finding, Western blot immunofluorescence experiments showed strong activation of the extrinsic apoptotic pathway, and RNAi knockdown experiments showed requirements for both caspase 8 and Rip1 in a synergistic response ( 14A-14G, 24E, and 24F). Furthermore, manipulation of TNFα to VSVΔ51 improved the synergy with SMC therapy by an order of magnitude (FIGS. 15A and 15B).

  Next, when type I IFN receptor (IFNAR1) was silenced, it was unexpectedly found that IFNAR1 knockdown prevented synergy between SMC therapy and oncolytic VSV (FIG. 3B, Figures 13B and 24D). Because TRAIL is an established ISG in response to type I IFN28, IFNAR1 knockdown was predicted to blunt but not completely suppress bystander killing. Although TNFα and IL-1β appear to be independent of IFN signaling, they nevertheless respond to NF-κB signaling downstream of viral detection. This result suggests the possibility of a non-standard type I IFN dependent pathway for the production of TNFα and / or IL-1β. In fact, when mRNA expression of IFNβ, TRAIL, TNFα, and IL-1β is scrutinized during oncolytic VSV infection, a significant temporal delay is found between the induction of IFNβ and the induction of TRAIL and TNFα. Was done (Figure 3C). This data also suggests that TNFα can be induced next to IFNβ, like TRAIL. To demonstrate this concept, IFNAR1 was silenced prior to treatment of cells with VSVΔ51. IFNAR1 knockdown completely abolished the induction of both TRAIL and TNFα by oncolytic VSV (FIG. 3D). Furthermore, the synergy with SMC was summarized using recombinant type I IFN (IFNα / β) and type II IFN (IFNγ) rather than type III IFN (IL28 / 29) (FIG. 3E). Taken together, these data indicate that type I IFN is required for the induction of TNFα and TRAIL during VSV Δ51 infection of tumor cells. Furthermore, the production of these cytokines is responsible for the bystander killing of neighboring, uninfected SMC-treated cells.

  To further investigate non-standard induction of TNFα, mRNA expression levels of TRAIL and TNFα were measured in SNB75 cells treated with recombinant IFNβ. Both cytokines were induced by IFNβ treatment (FIG. 3F) and ELISA experiments confirmed the production of the corresponding protein product in cell culture medium (FIG. 3G). Interestingly, there was a significant temporal delay between the induction of TRAIL and the induction of TNFα. As TRAIL is an authentic ISG and TNFα is not, this result has the potential to respond to downstream ISGs that are not directly induced by IFNβ but upregulated by IFNβ. Thus, quantitative RT-PCR was performed on the 176 cytokines in SNB75 cells, and 70 species significantly upregulated by IFNβ were identified (Table 5). The role of these ISGs in the induction of TNFα by IFNβ is currently being investigated. It is also interesting to note that SMC treatment promoted the induction of both TRAIL and TNFα by IFNβ in SNB75 cells (FIGS. 3F and 3G). Furthermore, using dominant negative constructs of IKK, it was found that the production of these inflammatory cytokines downstream of IFNβ is at least partially dependent on classical NF-κB signaling (FIG. 3H) . In EMT6 cells, SMC treatment was found to enhance cell production of TNFα (5 to 7-fold percentage increase) upon VSV infection (FIG. 16). Finally, blocking TNF-R1 signaling (with antibody or siRNA) was also shown to prevent EMT6 cell death in the presence of SMC and VSVΔ51 or IFNβ (FIGS. 17A-17C and 24H). The relationship between type I IFN and TNFα is complex and may have complementary or inhibitory effects depending on the biological context. However, without limiting the present invention to any particular mechanism of action, a simple working model can be proposed as follows: Tumor cells infected with oncolytic RNA virus have type I IFN up. Regulating, this process is not affected by SMC antagonism of IAP proteins. These IFNs then signal to nearby uninfected cancer cells to express and secrete TNFα and TRAIL, and this process is enhanced by SMC treatment, resulting in no exposure to SMCs. It induces autocrine and paracrine programmed cell death in infected tumor cells (FIGS. 18A and 18B).

Oncolytic VSV promotes SMC therapy in preclinical animal models of cancer
EMT 6 breast adenocarcinoma was used as a syngeneic model to evaluate concurrent SMC and oncolytic VSV therapy in vivo. Preliminary safety and pharmacodynamic experiments show that the 50 mg / kg dose of LCL 161 delivered by oral gavage is well tolerated, at least 24 hours, and in some cases up to 48 to 72 hours in the tumor. cIAP1 / 2 knockdown was induced (FIG. 19A, FIG. 19B, and FIG. 24G). When the tumors reached approximately 100 mm ³ we started treating mice twice weekly with SMC and VSV Δ51 and delivered systemically. As a single agent, SMC therapy resulted in a decrease in tumor growth rate and a moderate prolongation of survival, while VSV Δ51 treatment did not affect tumor size or survival (FIGS. 4A and 4B). In sharp contrast, combination treatment with SMC and VSVΔ51 induced dramatic tumor regression, resulting in permanent cure in 40% of the treated mice. Consistent with the bystander killing mechanism elucidated in vitro, immunofluorescence analysis shows that the infectivity of VSV Δ51 is transient and limited to small foci within the tumor (FIG. 4C), but the activity of caspase 3 is Was shown to spread to tumors co-treated with SMC and VSVΔ51 (FIG. 4D). In addition, immunoblots using tumor lysates showed activation of caspase 8 and caspase 3 in dual-treated tumors (FIG. 4E, FIG. 24B, and FIG. 24G). Animals in the combination treatment cohort experienced weight loss but mice recovered completely after the last treatment (FIG. 20A).

  To confirm these in vivo data in another model system, a human HT-29 colorectal adenocarcinoma xenograft model was tested in nude (athymic) mice. HT-29 is a cell line that responds highly to bystander killing by co-treatment of SMC and VSV Δ51 in vitro (FIGS. 21A and 21B). Similar to our findings in the EMT6 model system, combination therapy with SMC and VSVΔ51 induced tumor regression and a significant prolongation of mouse survival (FIG. 21C). In contrast, neither monotherapy had any effect on HT-29 tumors. Furthermore, there was no further weight loss in the dual-treated mice as compared to SMC-treated mice (FIG. 21D). These results indicate that synergy is very effective in the refractory xenograft model, and that the adaptive immune response does not initially have a major role in the efficacy of the SMC and OV co-therapy.

Role of innate antiviral response and immune effectors in co-treatment synergism Next, it was determined if oncolytic VSV infection in combination with SMC treatment resulted in TNFα or IFNβ mediated cell death in vivo. It was investigated whether blocking TNFα signaling by neutralizing antibodies affected the synergy of SMC and VSVΔ51 in the EMT6 tumor model. Application of TNFα neutralizing antibody reversed tumor regression and reduced the viability to values close to the control and single treatment groups as compared to the isotype matched antibody control (FIG. 4F and FIG. 4G). This indicates that TNFα is required in vivo for the combined antitumor efficacy of SMC and oncolytic VSV.

  In order to investigate the role of IFNβ signaling in the combination SMC and OV paradigm, Balb / c mice bearing EMT6 tumors were treated with IFNAR1 blocking antibodies. Mice treated with IFNAR1 blocking antibody succumbed to viremia within 24 to 48 hours post infection. Before death, tumors were harvested 18-20 hours after viral infection and tumors were analyzed for caspase activity. Although these animals deficient in type I IFN signaling were ill due to large viral load, excised tumors showed evidence of caspase 8 activity and the expected activity of caspases in the tumor In contrast to the control group (FIG. 22), only minimal signs of caspase 3 activity were shown (FIG. 22). These results support the hypothesis that intact type I IFN signaling is required to mediate the anti-tumor effects of the combined approach.

To evaluate the contribution of innate immune cells or other immune mediators to the efficacy of OV / SMC combination therapy, first treat EMT6 tumors in immune deficient NOD-skid or NSG (NOD-skid-IL2R gamma ^null ) mice I tried to. However, similar to the IFNAR1 depletion signaling study, these mice also died rapidly due to viremia. Thus, the contribution of innate immune cells was addressed by using an ex vivo splenocyte culture system as a surrogate model. Innate immune populations with the ability to produce TNFα were positively selected and further sorted from naive splenocytes. Macrophages (CD11b + F4 / 80 +), neutrophils (CD11b + Gr1 +), NK cells (CD11b-CD49b +) and bone marrow negative (lymphocytes) populations (CD11b-CD49-) are stimulated with VSV Δ51 and conditioned medium The cells were transferred to EMT6 cells and cytotoxicity was measured in the presence of SMC. These results indicate that VSV Δ51-stimulated macrophages and neutrophils, but not NK cells, can produce factors that lead to cancer cell death in the presence of SMC (FIG. 23A). Bone marrow derived primary macrophages were also isolated and these macrophages also produced factors that killed EMT6 cells in response to oncolytic VSV infection in a dose dependent manner (FIG. 23B). In summary, these findings indicate that multiple innate immune cell populations can respond to mediate the observed anti-tumor effects, and that macrophages are the most likely effectors of this response. ing.

The immune adjuvants poly (I: C) and CpG promote SMC therapy in vivo Next, do synthetic TLR agonists known to induce a congenital proinflammatory response synergize with SMC therapy? I investigated. EMT6 cells were co-cultured with mouse splenocytes in a transwell insertion system and splenocytes were treated with SMC and an agonist of TLR3, 4, 7 or 9. All tested TLR agonists were found to induce bystander cell death of EMT6 cells treated with SMC (FIG. 5A). The TLR4, 7 and 9 agonists LPS, imiquimod and CpG, respectively, probably cause splenocytes to induce bystander killing of EMT6 cells, as their target TLR receptors are not expressed in EMT6 cells I needed it. However, the TLR3 agonist poly (I: C) resulted in cell death of EMT6 cells directly in the presence of SMC. Next, poly (I: C) and CpG were tested in vivo in combination with SMC therapy. These agonists were chosen because they have been shown to be safe in humans and are currently in several mid-to-late clinical trials for cancer. EMT6 tumors were established and treated as described above. Poly (I: C) treatment did not affect tumor growth as a single agent, but the combination with SMC induced substantial tumor regression, 60% of treated mice when delivered intraperitoneally Provided a permanent cure (Figures 5B and 5C). Similarly, CpG monotherapy did not affect tumor size or survival but resulted in tumor regression and permanent cure in 88% of treated mice when combined with SMC therapy (FIG. 5D and FIG. 5E). Importantly, these combination therapies were well tolerated by the mice and their weight returned to pre-treatment levels soon after cessation of therapy (Figures 20B and 20C). Taken together with the oncolytic VSV results, this data shows that a series of clinically advanced innate immune adjuvants strongly and safely synergize with SMC therapy in vivo in several cancers It has been shown to induce tumor regression and permanent healing in treatment-refractory, aggressive mouse models.

(Example 2)
Inactivated viral particles, cancer vaccines, and stimulatory cytokines synergize with SMC to kill the tumor
Use of current cancer immunotherapies such as BCG (Bacillus Calmette-Guerin), recombinant interferon (eg IFNα), and recombinant tumor necrosis factor (eg TNFα used in limb perfusion), and of the immune system Recent clinical use of biologics (eg, blocking antibodies) against immune checkpoint inhibitors (such as anti-CTLA-4 and anti-PD-1 or PD-L1 monoclonal antibodies) to overcome tumor-mediated suppression is an effective treatment modality Emphasize the potential of 'cancer immunotherapy'. As shown in Example 1, we demonstrated the robust potential of non-viral immunostimulatory agents that synergize with SMC (FIG. 5). To further illustrate these studies, we also non-replicating Rhabdovirus, a UV-irradiated VSV particle that retains its infectivity and immunostimulatory properties without the ability to replicate and spread. The potential of SMCs to synergize with derived particles (referred to as NRRPs) was tested. To assess whether NRRP interacts directly with SMC, we co-processed various cancer cell lines, EMT6, DBT, and CT-2A with SMC and various levels of NRRP. Cell viability was assessed by Alamar Blue. We observed that NRRP synergizes with SMC in these cancer cell lines (FIG. 25A). To assess whether NRRP can induce a potent proinflammatory response, we used fractionated mouse splenocytes with NRRP (or synthetic CpG ODN 2216 as a positive control) Treated, cell culture supernatants were transferred to EMT6 cells in culture in a dose-response manner and cells were treated with vehicle or SMC. We observed that the immunogenicity of NRRP was at similar levels of CpG, as there was a significant proinflammatory response that resulted in a high degree of EMT6 cell death in the presence of SMC (FIG. 25B) ). Because CpG and SMC treatment in the EMT6 tumor model resulted in a cure rate of 88% (Figure 5D), these findings suggest that the combination of SMC and NRRP may be highly synergistic in vivo doing.

  Attenuation approved in practice by our success in the discovery of synergy between SMC and live or inactivated single-stranded RNA oncolytic rhabdovirus (eg, VSV Δ51, Maraba-MG1, and NRRP) It was suggested that the vaccine might be able to synergize with SMC. To test this possibility, we typically use the cancer biologic, tuberculosis mycobacterium, used to treat bladder cancer in situ due to high local production of TNFα. Aum, BCG's ability to synergize with SMC was assessed. In fact, the combination of SMC and BCG interacts strongly to kill EMT6 cells in vitro (FIG. 26A). These findings have also been extended to in vivo as well; we have administered oral SMC and BCG administered locally or systemically (ie, administered intratumorally or intraperitoneally, respectively) Significant tumor regression was observed for the combination treatment of (Figure 26B). These findings demonstrate the applicability of the approved vaccine for combination cancer immunotherapy with SMC.

Type I IFN synergizes with SMC in vivo The effects of viruses, and possibly other TLR agonists and vaccines, are controlled, in part, by type I IFN production, which is controlled by various signaling mechanisms such as mRNA translation. It is believed to be mediated. Our findings have elicited the different possibilities of combining SMC treatment with current immunotherapies, such as recombinant IFN, as an effective approach to treat cancer. To explore the potential of this combination, we conducted two treatment regimens, SMC and either intraperitoneal or intratumoral injection of recombinant IFNα in the syngeneic orthotopic EMT 6 breast adenocarcinoma model The While treatment with IFNα had no effect on EMT6 tumor growth or overall survival, SMC treatment slightly prolonged the survival of mice and had a 17% cure rate (FIG. 27). However, combined treatment with SMC and intraperitoneal or intratumoral injection of IFNα significantly delayed tumor growth and prolonged survival of tumor-bearing mice, resulting in 57% and 86% cure rates, respectively. (Figure 27). These results support the hypothesis that direct stimulation with type I IFN can synergize with SMC to eradicate tumors in vivo.

Further evaluation of the oncolytic rhabdovirus on the possibility of synergy with SMC
While VSVΔ51 is a preclinical candidate, the oncolytic rhabdoviruses VSV-IFNβ and Maraba-MG1 are currently undergoing clinical trials in cancer patients. As shown in Example 1, we demonstrated that Maraba-MG1 synergizes with SMC in vitro (FIG. 9). We also synergize SMC with the clinical candidates VSV-IFNβ and VSV-NIS-IFNβ (ie, carry the imaging gene, NIS, sodium-iodine cotransporter) in EMT6 cells I also confirmed that (Figure 28). To assess whether these viruses can induce a proinflammatory state in vivo, we infected 5 x 10 ⁸ PFU of VSV Δ51, VSV-IFNβ, and iv with infected mice. The levels of TNFα were measured from sera of Maraba-MG1 treated and infected mice. In all cases there was a transient but robust increase in TNFα from oncolytic virus infection at 12 hours post infection, which was barely detectable by 24 hours (Figure 29) . This is of course because these infections are self-limiting in immunocompetent hosts. These results suggest that the clinical candidate oncolytic Rhabdovirus has the potential to synergize with SMC in a manner similar to VSVΔ51.

As shown in Example 1, we show that a form of VSV Δ51 engineered to express full-length TNFα enhances oncolytic virus-induced cell death in the presence of SMC Demonstrated that it can be done (Figure 15). To further explain these findings in detail, we also used VSVΔ51 as a form of TNFα (VSVΔ51-solTNFα) whose intracellular and transmembrane components were replaced by a secretion signal derived from human serum albumin. It was engineered to express. While solTNF-α is constitutively secreted from host cells compared to full-length TNFα (memTNFα), the memTNFα form can be fixed on cell membranes (still can induce cell death in a contact secretion mode) Or may be released due to endogenous processing by metalloproteases (such as ADAM17) and kill cells in a paracrine manner. We assessed whether both forms of TNFα derived from oncolytic VSV infected cells synergize with SMC in the orthotopic syngeneic breast cancer model, EMT6. As expected, treatment with SMC slightly slows the EMT6 tumor growth rate and slightly prolongs the survival of tumor-bearing mice, and the combination of vehicle and either VSVΔ51-memTNFα or VSVΔ51-solTNFα is overall survival or It did not affect the tumor growth rate (Figures 30A and 30B). On the other hand, TNFα expressed by the virus significantly delayed the tumor growth rate, resulting in an increase in survival of 30% and 70%, respectively. Notably, the 40% tumor cure rate (FIG. 4A) derived from the combination of SMC and VSV Δ51 required 4 treatments and a dose of 5 × 10 ⁸ PFU of VSV Δ51. However, the combination of oncolytic VSV and SMC expressing TNFα resulted in a higher cure rate and was achieved using two treatment regimens with a viral dose of 1 × 10 ⁸ PFU. To evaluate whether this treatment strategy can be applied to other refractory syngeneic models, we use VSVΔ51-solTNFα in synergy with SMC in a subcutaneous model of mouse colon cancer cell line CT-26. It evaluated whether it acted. As expected, we observed a modest reduction in tumor growth rate and a slight prolongation of survival without observing the effect of tumor growth rate or survival on VSV Δ51-solTNFα (FIG. 30C). However, we were able to further delay the tumor growth and prolong the survival of these tumor-bearing mice using a combination treatment of SMC and VSVΔ51-solTNFα. Thus, the inclusion of the TNFα transgene in the oncolytic virus is a major advantage for the SMC combination. The inclusion in the virus of other cell death ligand transgenes, such as TRAIL, FasL, or lymphotoxin, to synergize with SMC can be easily envisioned.

Search for the potential of SMC to eradicate brain tumors
The combination of SMC and immunostimulatory agents is applicable to many different types of cancer, such as brain malignancy where there is no effective therapy and immunotherapy can be expected. As the first step, since the blood-brain barrier (BBB) is a major barrier to drugs entering the brain, we determined whether SMC can cross the BBB in mouse models of brain tumors. . We show that SMC-induced degradation of cIAP1 / 2 protein in intracranial CT-2A tumors several hours after drug administration causes SMCs to cross the BBB, cIAP1 / 2 in brain tumors and potentially Were observed to show that it can antagonize XIAP (FIG. 31A). We also found that direct intracranial injection of SMC (10 μL of a 100 μM solution) can result in strong downregulation of both cIAP1 / 2 and XIAP proteins (FIG. 31B), It was shown to be a direct consequence of SMC-induced autoubiquitination of IAP or, in the case of XIAP loss, a consequence of tumor cell death induction. As a second step, we wanted to determine if systemic stimulation of immunostimulatory agents could result in a proinflammatory response in the brains of naive mice. Indeed, we observed a marked upregulation of TNFα levels from brains from mice injected intraperitoneally with the virus mimic, poly (I: C), TLR3 agonist (FIG. 32A). We have extracted crude protein lysates from brains of mice treated with poly (I: C) or clinical candidate oncolytic rhabdovirus VSVΔ51, VSV-IFNβ, or Maraba-MG1. This finding was followed up by applying these lysates to CT-2A or K1580 glioblastoma cells in the presence of SMC. We observe that stimulation of the innate immune response with these nonviral synthetic or bioviral agents results in enhanced cell death in the presence of SMC for these two glioblastoma cell lines (FIG. 32B). As a third step, we also confirmed that poly (I: C) can be directly administered intracranially without apparent toxicity, which may even result in increased cytokine induction at the site of the tumor ( Figure 32C). Finally, we assessed whether direct immune stimulation within brain or whole body stimulation results in permanent cure in SMC-treated mouse models of brain cancer. Combination of SMC (oral) and poly (I: C) (intracranial) or VSV Δ51 (iv) results in almost complete survival of glioma in CT-2A bearing mice (Figures 32D and 32E). The expected survival rates are 86% and 100%, respectively. As a follow-up to the observed synergy of SMC and intracranial treatment of poly (I: C), we also direct, simultaneous the SMC and recombinant human IFNα (B / D). The potential for the treatment of CT-2A glioma with intracranial injection was evaluated. In fact, we observed a significant positive impact of mouse survival when using the combination treatment, with a cure rate of 50% (Figure 33). Importantly, single or combined SMC or IFNα treatment did not result in overt neurotoxicity in these tumor-bearing mice. Overall, these results indicate that multiple modes of SMC treatment synergize with multiple locally or systemically administered innate immune stimulants to kill cancer cells in vitro and cause cancer Show that tumors can be eradicated in animal models of

Method Reagent
Novartis provided LCL 161 (Houghton, PJ, et al., Initial testing (stage 1) of LCL 161, a SMAC mimetic, by the Pediatric Preclinical Testing Program. Pediatr Blood Cancer 58: 636-639 (2012); Chen, KF Et al, Inhibition of Bcl-2 improves effect of LCL 161, a SMAC mimetic, in hepatocellular carcinoma cells. Biochemical Pharmacology 84: 268-277 (2012)). SM-122 and SM-164 are Dr. Shaomeng Wang (University of Michigan, USA) (Sun, H. et al., Design, synthesis, and characterization of a potent, nonpeptide, cellpermeable, bivalent Smac mimetic that simultaneously targets both the BIR2 and BIR3 domains in XIAP. J Am Chem Soc 129: 15279-15294 (2007)). AEG 40 730 (Bertrand, MJ et al., CIAP1 and cIAP2 facilitate cancer cell survival by functioning as E3 ligases that promote RIP1 ubiquitination. MoI Cell 30: 689-700 (2008)) was synthesized by Vibrant Pharma Inc (Brantford, Canada) The OICR 720 was synthesized by the Ontario Institute for Cancer Research (Toronto, Canada) (Enwere, EK et al., TWEAK and cIAP1 regulated myoblast fusion through the noncanonical NF- kappa B signaling pathway. Sci Signal 5: ra75 (2013)). IFNα, IFNβ, IL28 and IL29 were obtained from PBL Interferonsource (Piscataway, USA). All siRNAs were obtained from Dharmacon (Ottawa, Canada; ON TARGET plus SMARTpool). CpG-ODN 2216 was synthesized by IDT (5'-gggGGACGATCGTCCgggggg-3 '(SEQ ID NO: 1), lower case letters indicate phosphorothioate linkages between these nucleotides, while italics are three, including phosphodiester bonds Identify CpG motifs). Imiquimod was purchased from BioVision Inc. (Milpitas, USA). Poly (I: C) was obtained from InvivoGen (San Diego, USA). LPS was from Sigma (Oakville, Canada).

Cell Culture Cells were maintained at 37 ° C. and 5% CO 2 in DMEM medium supplemented with 10% heat-inactivated fetal bovine serum, penicillin, streptomycin and 1% non-essential amino acids (Invitrogen, Burlington, USA). All cell lines were obtained from the ATCC except for the following: SNB75 (Dr. D. Stojdl, Children's Hospital of Eastern Ontario Research Institute) and SF539 (UCSF Brain Tumor Bank). Cell lines were regularly tested for mycoplasma contamination. For siRNA transfection, cells were reverse transfected with Lipofectamine RNAiMAX (Invitrogen) or DharmaFECT I (Dharmacon) for 48 hours according to the manufacturer's protocol.

Virus
In this test, VSV Δ51 of Indiana serotype (Stojdl, DF et al., VSV strains with defects in their ability to shutdown innate immunity are potent systemic anti-cancer agents. Cancer Cell 4 (4), pp. 263-275 (2003)) Used and grown in Vero cells. VSVΔ51-GFP is a recombinant derivative of VSVΔ51 that expresses the jellyfish green fluorescent protein. VSVΔ51-Fluc expresses firefly luciferase. VSVΔ51 (VSVΔ51ΔG) lacking the gene encoding the glycoprotein was grown in pMD2-G transfected HEK293T cells using Lipofectamine 2000 (Invitrogen). The full-length human TNFα gene was inserted between the G and L virus genes to generate a VSV Δ51-TNFα construct. All VSV Δ51 viruses were purified on sucrose cushions. Maraba-MG1, VVDD-B18R-, Reovirus and HSV1 ICP34.5 were generated as previously described (Brun, J. et al., Identification of genetically modified Maraba virus as an oncolytic rhabdovirus. MoI Ther 18, 1440 Le Boeuf, F. et al., Synergistic interaction between oncolytic viruses augments tumor killing. MoI Ther 18, pp. 888-895 (2011); Lun, X. et al., Efficacy and safety / toxicity study of recombinant vaccinia. virus JX-594 in two immunocompetent animal models of glioma. Mol Ther 18, pages 1927 to 1936 (2010)). Generation of adenoviral vectors expressing GFP or coexpressing GFP and dominant negative IKKβ was as previously described.

In Vitro Viability Assay Cell lines were seeded in 96 well plates and incubated overnight. Cells are treated with vehicle (0.05% DMSO) or 5 μM LCL 161 and infected with OV at the indicated MOI or 250 U / mL IFN beta, 500 U / mL IFN alpha, 500 U / mL IFN gamma, 10 ng / mL The cells were treated with IL28 of 10 or 48 ng of IL29 for 10 hours. Cell viability was determined by alamar blue (resazurin sodium salt (Sigma)) and data normalized to vehicle treatment. The sample size chosen is consistent with previous reports that used similar analysis for viability assay. For combination factor, cells are seeded overnight and treated for 48 hours with serial dilutions of fixed mixing ratios of VSV Δ51 and LCL 161 (5000: 1, 1000: 1 and 400: 1 ratio PFU VSV Δ51: μM LCL161) , Cell viability was assessed by Alamar Blue. Combination coefficients (CI) were calculated according to the method of Chou and Talalay using Calcusyn (Chou, TC & Talaly, P. A simple generalized equations for the analysis of multiple inhibitions of Michaelis-Menten kinetics systems. J Biol Chem 252, 6438-6442 (1977)). Statistical measures (mean and standard deviation or standard error) were determined using n = 3 biological replicates.

Diffusion assay
Confluent monolayers of 786-0 cells were covered with 0.7% agarose in complete medium. A small hole was pipetted into the agarose overlay in the middle of the well, to which 5 × 10 ³ PFU of VSV Δ51-GFP was administered. Vehicle or media containing 5 μM LCL161 was added to the top of the overlay, cells were incubated for 4 days, fluorescence images were acquired, and cells were stained with crystal violet.

Splenocyte co-culture
EMT6 cells were cultured in multiwell plates and covered with cell culture inserts containing unfractionated splenocytes. Briefly, single cell suspensions were obtained by passing mouse spleen through a 70 μm nylon mesh and red blood cells were lysed with ACK lysis buffer. Before placing splenocytes in the presence of 1 μM LCL 161, either with 0.1 MOI VSV Δ51ΔG, 1 μg / mL poly (I: C), 1 μg / mL LPS, 2 μM imiquimod, or 0.25 μM CpG Processed for 24 hours. Viability of EMT6 cells was determined by crystal violet staining. Statistical measures (mean, standard deviation) were determined using n = 3 biological replicates.

Cytokine Responsive Bioassay Cells were infected with VSV Δ51 at the indicated MOI for 24 hours and cell culture supernatants were exposed to UV light for 1 hour to inactivate the VSV Δ51 particles. Subsequently, the UV inactivated supernatant was added to naive cells for 48 hours in the presence of 5 μM LCL161. Cell viability was assessed by Alamar Blue. Statistical measures (mean, standard deviation) were determined using n = 3 biological replicates.

Microscopy To measure caspase 3/7 activation, 5 μM LCL 161, indicated MOI VSV Δ 51, and 5 μM CellPlayer Apoptosis Caspase 3/7 reagent (Essen Bioscience, Ann Arbor, USA) were added to the cells . Cells were placed in an incubator equipped with an IncuCyte Zoom microscope equipped with a 10 × objective and phase contrast and fluorescence images were acquired over a 48 hour span. Alternatively, cells were treated with 5 μM LCL 161 and 0.1 MOI of VSV Δ51-GFP and SMC for 36 hours and labeled with Magic Red Caspase-3 / 7 Assay Kit (ImmunoChemistry Technologies, Bloomington, USA). To measure the percentage of apoptotic cells, 1 μg / mL Annexin V-CF 594 (Biotium, Hayward, USA) and 0.2 μM YOYO-1 (Invitrogen) were added to SMC and VSV Δ51 treated cells. Images were acquired 24 hours after treatment using the IncuCyte Zoom. Fluorescent signal counts were processed using the integrated object counting algorithm in IncuCyte Zoom software. Statistical measures (mean, standard deviation) were determined using biological replicates of n = 12 (caspase 3/7) or n = 9 (Annexin V, YOYO-1).

Multi-Step Growth Curve Cells were treated with vehicle or 5 μM LCL 161 for 2 hours and then infected with VSV Δ51 at the indicated MOI for 1 hour. The cells were washed with PBS and the cells were supplemented with vehicle or 5 μM LCL161 and incubated at 37 ° C. Aliquots were taken at indicated times and virus titer was assessed by standard plaque assay using African green monkey VERO cells.

Western immunoblotting Cells were scraped off, harvested by centrifugation and lysed in RIPA lysis buffer containing a protease inhibitor cocktail (Roche, Laval, Canada). Equal amounts of soluble proteins were separated on polyacrylamide gels and transferred to nitrocellulose membranes. The individual proteins were obtained from the following antibodies: pSTAT1 (9171), Caspase-3 (9661), Caspase-8 (9746), Caspase-9 (9508), DR5 (3696) from Cell Signaling Technology (Danvers, USA) , TNF-R1 (3736), cFLIP (3210), and PARP (9541); Caspase-8 from Enzo Life Sciences (Farmingdale, USA) (1612); IFNAR1 from Abcam (Cambridge, USA) (EP 899) And TNF-R1 (19139); Caspase-8 from Invitrogen (AHZ0502); cFLIP from Alexis Biochemicals (Lausen, Switzerland) (clone NF6); RIP1 from BD Biosciences (Franklin Lakes, USA) (clone 38) As well as by Western immunoblotting using E7 from Developmental Studies Hybridoma Bank (Iowa City, USA). Our rabbit anti-rat IAP1 and IAP3 polyclonal antibodies were used to detect human and mouse cIAP1 / 2 and XIAP, respectively. Primary antibodies were detected using AlexaFluor 680 (Invitrogen) or IRDye 800 (Li-Cor, Lincoln, USA) and infrared fluorescence signals were detected using the Odyssey Infrared Imaging System (Li-Cor).

RT-qPCR
Total RNA was isolated from cells using the RNAEasy Mini Plus kit (Qiagen, Toronto, Canada). Two step RT-qPCR was performed on Mastercycler ep realplex (Eppendorf, Mississauga, Canada) using Superscript III (Invitrogen) and SsoAdvanced SYBR Green supermix (BioRad, Mississauga, Canada). All primers were obtained from realtimeprimers.com. Statistical measures (mean, standard deviation) were determined using n = 3 biological replicates.

ELISA
Cells were infected with virus at the indicated MOI or treated with IFNβ for 24 hours and clarified cell culture supernatants were concentrated using an Amicon ultrafiltration unit. Cytokines were measured using the TNFα Quantikine high sensitivity, TNFα DuoSet, TRAIL DuoSet (R & D Systems, Minneapolis, USA) and VeriKine IFNβ (PBL Interferonsource) assay kits. Statistical analysis was determined using n = 3 biological replicates.

EMT 6 mammary tumor model
A mammary tumor was established by injecting 1 × 10 ⁵ wild type EMT6 or firefly luciferase tagged EMT6 (EMT6-Fluc) cells into the mammary fat pad of 6 week old female BALB / c mice. Mice with palpable tumors (about 100 mm ³ ) were treated with vehicle (30% 0.1 M HCl, 70% 0.1 M NaOAc, pH 4.63) or 50 mg / kg LCL 161 (oral) and PBS or 5 × The iv injection of 10 ⁸ PFU of VSV Δ51 was co-treated twice weekly for two weeks. Animals are treated twice weekly with LCL 161 for poly (I: C) 25 and SMC treatment and BSA (it), 20 μg poly (I: C) (it) or 2.5 mg / kg poly ( I: C) (ip) treated 4 times a week. The SMC and CpG groups were injected with 2 mg / kg CpG (ip) and the next day, CpG and SMC treatment was performed. CpG and SMC treatment was repeated 4 days later. Treatment groups were assigned per cage, each group having a minimum of n = 4-8 for statistical measures (mean, standard error; Kaplan-Meier with log rank analysis). The sample size is consistent with previous reports of examining tumor growth and mouse survival after cancer treatment. Blinding was impossible. Animals were euthanized when tumors metastasized intraperitoneally or tumor burden exceeded 2000 mm ³ . Tumor volume was calculated using (π) (W) ² (L) / 4, where W = the width of the tumor and L = the length of the tumor. Bioluminescent imaging of tumors was captured after ip injection of 4 mg of luciferin (Gold Biotechnology, St. Louis, USA), using a Xenogen 2000 IVIS CCD-camera system (Caliper Life Sciences, Mass., USA).

HT-29 subcutaneous tumor model
Subcutaneous tumors were established by injecting 3 × 10 ⁶ HT-29 cells into the right flank of 6 week old female CD-1 nude mice. Palpable tumors (about 200 mm ³ ) were treated with 5 intratumoral injections (it) of PBS or 1 × 10 ⁸ PFU of VSV Δ51. Four hours later, mice were orally administered vehicle or 50 mg / kg of LCL161. Treatment groups were assigned per cage, with each group having a minimum of n = 5-7 for statistical measures (mean, standard error; Kaplan-Meier using log rank analysis). The sample size is consistent with previous reports of examining tumor growth and mouse survival after cancer treatment. Blinding was impossible. The animals were euthanized when the tumor burden exceeded 2000 mm ³ . Tumor volume was calculated using (π) (W) ² (L) / 4, where W = the width of the tumor and L = the length of the tumor.

  All animal experiments were conducted with the approval of the University of Ottawa Animal Care and Veterinary Service, according to the guidelines established by the Canadian Council on Animal Care.

Antibody-mediated cytokine neutralization
To neutralize TNFα signaling in vitro, add 25 μg / mL α-TNFα (XT 3.11) or isotype control (HRNP) to EMT 6 cells for 1 hour, then with LCL 161 and VSV Δ51 or IFN β It processed simultaneously for 48 hours. Viability was assessed by Alamar Blue. To neutralize TNFα in the EMT6-Fluc tumor model, 0.5 mg of α-TNFα or α-HRNP was administered 8, 10 and 12 days after implantation. Mice were treated with 50 mg / kg LCL 161 (po) at 8, 10 and 12 days after implantation and infected with 5 × 10 ⁸ PFU of VSV Δ51 on days 9, 11 and 13 by iv. For neutralization of type I IFN signaling, 2.5 mg of α-IFNAR1 (MAR1-5A3) or isotype control (MOPC-21) is injected into EMT6 tumor-bearing mice, at 50 mg / kg of LCL 161 (po) Treated for 20 hours. Mice were infected with 5 × 10 ⁸ PFU of VSV Δ51 (iv) for 18-20 hours and tumors were processed for western blotting. All antibodies were from BioXCell (West Lebanon, USA).

Flow cytometry and sorting
EMT6 cells were co-treated with 0.1 MOI of VSV Δ51-GFP and 5 μM LCL 161 for 20 hours. Cells were trypsinized, permeabilized using a CytoFix / CytoPerm kit (BD Biosciences) and stained with APC-TNFα (MP6-XT22) (BD Biosciences). Cells were analyzed on a Cyan ADP 9 flow cytometer (Beckman Coulter, Mississauga, Canada) and data were analyzed using FlowJo (Tree Star, Ashland, USA).

  Splenocytes were enriched for CD11b using the EasySep CD11b positive selection kit (StemCell Technologies, Inc., Vancouver, Canada). CD49 + cells were enriched from the CD11b- fraction using the EasySep CD49b positive selection kit (StemCell Technologies). CD11b + cells were stained with F4 / 80-PE-Cy5 (BM8, eBioscience) and Gr1-FITC (RB6-8C5, BD Biosciences) and further sorted using MoFlo Astroios (Beckman Coulter). Flow cytometry data were analyzed using Kaluza (Beckman Coulter). The isolated cells were infected with VSVΔ51 for 24 hours, and the clarified cell culture supernatant was added to EMT6 cells for 24 hours in the presence of 5 μM LCL161.

Bone marrow derived macrophages Mouse femurs and ribs were removed and washed away to remove bone marrow. The cells were cultured in RPMI containing 8% FBS and 5 ng / ml M-CSF for 7 days. Flow cytometry was used to confirm the purity of the macrophages (F4 / 80 + CD11b +).

Immunohistochemistry Excised tumors were fixed in 4% PFA, embedded in a 1: 1 mixture of OCT compound and 30% sucrose and sectioned on a cryostat at 12 μm. Sections were permeabilized with 0.1% Triton X-100 in blocking solution (50 mM Tris-HCl pH 7.4, 100 mM L-lysine, 145 mM NaCl and 1% BSA, 10% goat serum). After overnight incubation of α-cleaved caspase 3 (C92-605, BD Pharmingen, Mississauga, Canada) and polyclonal antiserum VSV (Dr. Earl Brown, University of Ottawa, Canada), AlexaFluor-conjugated secondary antibody ( Secondary incubation with Invitrogen).

Statistical Analysis Kaplan-Meier survival plot comparisons were performed by log rank analysis, and then pairwise multiple comparisons were performed using the Holm-Sidak method (SigmaPlot, San Jose, USA). Calculation of EC ₅₀ values was performed in GraphPad Prism using normalized non-linear regression analysis. The EC ₅₀ shift was calculated by subtracting the log ₁₀ EC ₅₀ of SMC treated and VSV Δ51 infected cells from the log ₁₀ EC ₅₀ of vehicle treated cells infected with VSV Δ51. In order to normalize the degree of SMC synergy, EC ₅₀ values were normalized to 100% to offset cell death induced by SMC treatment alone.

(Example 3)
SMC-Containing Immunotherapy Shows Antimyeloma Activity Immune Checkpoint Blocker Synergizes with SMC Treatment to Delay Disease Progression in MM Intravenous injection of MPC-11 cells stably expressing the luciferase transgene Embedded in the BALB / c mouse. This in vivo MM model mimics human disease well and follows predictable disease progression. MPC-11 cells are obtained from mouse plasmacytomas. After two rounds of treatment with monoclonal antibodies against SMC and PD-1 or CTLA-4, only anti-PD-1 antibody based treatments showed responses with respect to delaying disease progression. Mice treated with the combination of anti-PD-1 antibody and SMC showed the best response, with almost no tumor burden as determined by the luminescent signal (FIG. 35). This combination also significantly prolonged the survival of mice compared to the control group (p = 0.01) and also to PD-1 treatment alone (p = 0.0163).

Type 1 interferon synergizes with SMC to cause MM cell death
In vitro studies testing the effects of various cytokines in combination with SMC highlighted the potential of type 1 IFN. Specifically, IFNα and IFNβ showed very potent synergistic killing of MM cells with SMC in many of the cell lines tested (FIG. 36A). Using the same MPC-11 mouse model, mice were treated with recombinant IFNα and SMC at three different time points (FIG. 37).

Oncolytic virus synergizes with SMC to cause MM cell death An oncolytic virus derived from the varicella stomatitis virus, VSVΔ51, works well in synergy with SMC in vitro to generate cells in MPC-11 cells Causes death (Figures 36B and 36C). Combinations containing SMCs were also tested in the MPC-11 syngeneic mouse model. Combination treatment did not reduce tumor burden as effectively as VSVΔ51 alone and was not well tolerated by mice. Treatment with VSVΔ51 alone delayed disease progression, but the increase in survival was significant compared to the untreated control group (p = 0.0379, log rank analysis) (FIG. 38).

SMC synergizes with standard MM therapeutics
In vitro viability assay showed synergistic cell killing of MM cells in combination with SMC-based combination with the synthetic glucocorticoid, dexamethasone (Dex) (FIG. 39B). When SMC is combined with the glucocorticoid receptor antagonist RU 486, comparable levels of cell death are present and synergy is not due to GCR activation but rather due to its inhibitory effect on NF-κB Suggests that.

Combination treatment based on SMC activates NF-κB signaling and causes apoptosis in MM cells
SMC treatment effectively induced rapid degradation of cIAP1 and cIAP2 (FIG. 40A). As a single agent, SMC treatment increased NF-κB signaling; starting with a slight short-term boost in the classical pathway and then decreasing long as shown by the higher ratio of phosphorylated p65 to p65 (Figure 40B). As activation of the classical pathway diminished, the alternative NF-κB pathway was very strongly activated, as shown by the increased ratio of p52 to p100 (FIG. 40C). Apoptosis in cells was confirmed by the presence of cleaved poly (ADP-ribose) polymerase (PARP). Cleavage of PARP is often used as an apoptotic marker as it is a substrate for caspases in early stages of apoptosis.

  The combination of SMC and IFNβ (FIG. 41) or VSVΔ5 or VSVmIFN (containing the inserted gene for mouse IFNβ) (FIG. 42) had many of the same features as SMC treatment alone. For example, the classical pathway was finally downregulated and the alternative pathway was upregulated. There was also apoptosis, as shown by both PARP cleavage and caspase 8 cleavage. The IFN receptor, IFNAR1, was also downregulated with IFN treatment, but is of interest as it is required for a continuous response to IFNβ. When VSV treatment was used, RIP1 was almost completely degraded at late time points; this is yet another signal of apoptosis, as it is degraded by caspase 8 after lipoptosome formation.

Sensitivity to SMC in MM1R and MM1S is associated with glucocorticoid receptor expression
The responsiveness to SMC-mediated cell death changes dramatically between related human MM cell lines MM1R and MM1S, which are derived from the same parent cell line and differ only in the expression of GCR. MM1R (FIG. 39A), which showed no detectable expression of GCR, is very sensitive to SMC (FIG. 39C), but MM1S with high GCR expression is resistant. MM1S can be sensitive to SMC treatment when treated with Dex, or the GCR antagonist RU 486 (FIG. 39B).

Innate Immunostimulatory Upregulates Inhibitors of Adaptive Immune Response The human MM cell lines U266, MM1R and MM1S potently upregulated PD-L1 in response to IFNβ treatment. Comparable upregulation was seen for the combination of SMC and IFNβ. Another ligand for PD-1, PD-L2, was similarly upregulated with IFNβ based treatment. This effect was pronounced at both the early and late time points for both proteins (Figure 43). This suggests that any immunostimulatory agent that activates type I IFN results in the upregulation of T cell co-inhibitor molecules.

Combination of SMC and immunomodulators results in cancer cell death that also contains CD8 + T cells. Figures 44A and 44B reinjected EMT6 cells into the mammary fat pad of dual-treated, cured mice Data from the day of implantation) or CT-2A cells reinjected intracranially (from day of first implantation to 190 days). FIG. 44C shows data from experiments in which CT-2A glioma or EMT6 breast cancer cells were trypsinized, surface stained with conjugated isotype control IgG or anti-PD-L1 and processed for flow cytometry. Is a graph showing FIG. 44D shows that CD8 + T cells are enriched from splenocytes (naive mice or mice to which EMT6 tumors have previously cured) using CD8 T cell positive magnetic selection kit and ELISpot for detection of IFNγ and granzyme B. FIG. 5 is a graph showing data from an experiment that has been assayed. CD8 + T cells were cocultured with culture medium or cancer cells (the ratio of cancer cells to CD8 + T cells 12: 1) and 10 mg of control IgG or anti-PD-1 for 48 hours. Three mice were used as independent biological replicates (previously cured of EMT6 tumors). Since 4T1 and EMT6 cells carry the same major histocompatibility antigens, 4T1 cells were used as a negative control.

SMC synergizes with immune checkpoint inhibitors in an orthotopic mouse model of cancer Figure 45A shows that mice bearing EMT 6 mammary tumors are treated with PBS or 1 x 10 ⁸ PFU of VSVD 51 within the tumor 1 FIG. 6 is a graph showing data from treatment of the rounds and after 5 days mice were treated with vehicle or 50 mg / kg LCL 161 (SMC) (oral) in combination with 250 mg anti-PD (intraperitoneal (ip)). 45B and 45C show that mice bearing intracranial CT-2A or GL261 tumors were treated with vehicle or 75 mg / kg LCL 161 (oral) and 250 mg (ip) control IgG, anti-PD-1 or anti-CTLA-4. It is a graph which shows the data processed 4 times. FIG. 45D is a graph showing data from athymic CD-1 nude mice bearing CT-2A intracranial tumors treated with 75 mg / kg LCL 161 (oral) and 250 mg (ip) anti-PD-1.

(Example 4)
Smac mimetics synergize with immune checkpoint inhibitors to promote tumor immunity
RPMI-8226, U266, MM1R, MM1S, MPC-11 cell lines were obtained from ATCC. MPC-11 is cultured in DMEM (Hyclone) containing 10% FBS (Hyclone), U266 is cultured in RPMI-1640 (Hyclone) containing 15% FBS, and all other cell lines Were cultured in RPMI-1640 containing 10% FBS.

  Cells were maintained at 37 ° C. and 5% CO 2 in DMEM medium supplemented with 10% heat-inactivated fetal calf serum and 1% non-essential amino acids (Invitrogen). All cell lines were obtained from ATCC except for: SNB 75 (Dr. D. Stojdl, Children's Hospital of Eastern Ontario Research Institute) and SF539 (UCSF Brain Tumor Bank). Primary NF1-/ + p53-/ + cells were from C57B1 / 6J p53 + / − / NF1 +/− mice. Cell lines were regularly tested for mycoplasma contamination. BTIC was cultured in serum-free culture medium supplemented with EGF and FGF-250. For siRNA transfection, cells were reverse transfected with Lipofectamine RNAiMAX (Invitrogen) for 48 hours according to the manufacturer's protocol. Cell lines were regularly tested for mycoplasma contamination. BTIC was cultured in serum-free culture medium supplemented with EGF and FGF-2. For siRNA transfection, cells were reverse transfected with Lipofectamine RNAiMAX (Invitrogen) for 48 hours according to the manufacturer's protocol.

Antibody and reagent
In vivo: LCL 161 was a generous gift from Novartis. Anti-PD-1 (clone J43) was purchased from BioXcell. Poly (I: C) (HMW vaccine grade, Invivogen). IFNα (for in vivo use) was a generous gift from Dr. Peter Staeheli of Germany. Billinapant was obtained from Tetralogic Pharmaceuticals.

  In vitro: IFN was obtained from PBL assay science. Dexamethasone and RU 486 were purchased from Sigma Aldrich.

  The antibodies used were RIAP1 (in-house), PD-L1 (Abcam), PD-L2 (R & D Systems), GCR (Santa Cruz), P100 (Cell Signaling), P65 (cell signaling), p- P65 (cell signalling), IFNAR1 (Abcam), PARP (Cell Signaling), tubulin (Developmental Studies Hybridoma Bank), RIP 1 (R & D Systems), caspase 8 (R & D Systems).

  AT-406, GDC-0917, and AZD-5582 were purchased from Active Biochem. TNF-α was purchased from Enzo. IFN-β was obtained from PBL Assay Science. The broad spectrum host IFN-αB / D was produced in yeast and purified by affinity chromatography. Non-targeting siRNA or siRNA targeting cFLIP was obtained from Dharmacon (ON-TARGET plus SMART pool). High molecular weight poly (I: C) was obtained from Invivogen.

Animal experimentation
Four to five week old BALB / c mice were purchased from Charles River and injected IV with 1 × 10 ⁶ MPC-11 Fluc cells stably expressing the firefly luciferase (Fluc) transgene. The treatment comprises 50 mg / kg LCL 161, 250 μg anti-PD-1 antibody, 250 μg anti-CTLA-4 antibody, 25 μg poly (I: C), 5 × 10 ⁸ pfu VSV Δ51, 1 μg IFNα. After IP injection of 200 μL of luciferin to measure luminescence, imaging of mice was performed using the in vivo imaging system IVIS.

Virus
Indiana serotype VSV was used in this study. VSV-EGFP, VSVΔ51 (lacking amino acid 51 in the M gene) and Maraba-MG1 were grown in Vero cells and purified on OptiPrep gradients. VSVΔ51 (VSVΔ51ΔG) lacking the gene encoding the glycoprotein was grown in pMD2-G transfected HEK293T cells using Lipofectamine 2000 (Invitrogen) and purified on sucrose cushions. NRRP was generated by exposing VSV-EGFP to UV (250 mJ cm-2) using a XL-1000 UV crosslinker (Spectrolinker).

In Vitro Viability Assay Cell lines were seeded in 96 well plates and incubated overnight. Cells were treated with vehicle (0.05% DMSO) or LCL 161 and infected with the indicated MOI virus, or 1 μg / mL IFN-α B / D, 0.1 ng / mL TNF-α, or indicated NRRP Processed for 48 hours. Cell viability was determined by alamar blue (resazurin sodium salt (Sigma)) and data normalized to vehicle treatment. Although sample methods were not determined using statistical methods, the sample sizes chosen are consistent with previous reports using similar analysis for viability assay.

Western Blotting Cells were scraped off, harvested by centrifugation and lysed in RIPA lysis buffer containing protease inhibitor cocktail (Roche). Tumors were excised, minced and lysed as above. Equal amounts of soluble protein were transferred to nitrocellulose membrane after separation on polyacrylamide gel. Individual proteins were detected by Western blotting using cFLIP (7F10, 1: 500, Alexis Biochemicals) and β-tubulin (1: 1000, E7, Developmental Studies Hybridoma Bank). Human and mouse cIAP1 / 2 and XIAP were detected using rabbit anti-rat IAP1 and IAP3 polyclonal antibodies, respectively (1: 5000, Cyclex Co.). Primary antibodies were detected using AlexaFluor 680 (Invitrogen) or IRDye 800 (Li-Cor) (1: 2500) and infrared fluorescence signals were detected using the Odyssey Infrared Imaging System (Li-Cor). Full length blots are shown in FIGS. 68A-68D.

ELISA
For in vivo detection of TNF-α, mice were treated intraperitoneally (ip) with 50 μg of poly (I: C) or intravenously (iv) with 5 × 10 ⁸ PFU of VSV Δ51. Brains were homogenized in 20 mM HEPES-KOH (pH 7.4), 150 mM NaCl, 10% glycerol and 1 mM MgCl 2 with the addition of EDTA-free protease inhibitor cocktail (Roche). NP-40 was added to a final concentration of 0.1% and clarified by centrifugation. Equal amounts were processed for detection of TNF-α using the TNF-α Quantikine assay kit (R & D Systems).

1 x 10 ⁶ mice with CT-2A tumor cured by treatment with SMC and anti-PD-1 antibody and age matched controls (naive) C57BL / 6 female mice to assess the specificity of the adaptive immune response CT-2A cells were injected subcutaneously. After 7 days, splenocytes were isolated and co-cultured with CT-2A cells for 48 hours in the presence of vehicle or 5 μM SMC or 20 μg / mL indicated antibody (ratio of spleen cells to cancer cells 20: 1 ). Secretion of IFN-γ, GrzB, TNF-α, IL-17, IL-6, and IL-10 was determined by ELISA (kit is from R & D Systems).

CT-2A and GL261 Brain Tumor Models Female 5 week old C57BL / 6 or CD-1 nude mice were anesthetized with isoflurane, shaved at the surgical site and prepared with 70% ethanol. 5 × 10 ⁴ cells were stereotactically injected at a volume of 10 μL into the left striatum over the course of one minute in the following coordinates: 0.5 mm anterior, 2 mm lateral from bregma, and 3.5 mm deep. The skin was closed using a surgical adhesive. Mice were orally treated with vehicle (30% 0.1 M HCl, 70% 0.1 M NaOAc, pH 4.63) or 75 mg / kg LCL 161 orally with 50 μg poly (I: C) in 10 μL (it's within the tumor) , 5 × 10 ⁸ VSV Δ 51 intravenously (iv) or 250 μg of anti-CD4 antibody (GK 1.5), anti-CD8 antibody (YTS 169.4), anti-PD1 antibody (J43), or CTLA-4 (9H10) Treated intraperitoneally (ip) with

  For treatment with birinapant, mice were treated with vehicle (12.5% Captisol) or 30 mg / kg birinapant (ip). In some cases, animals were treated with anti-IFNAR1 antibody (MAR1-5A3), anti-IFN-γ antibody (R4-6A2) or anti-TNF-α antibody (XT3.11). Isotype control IgG antibodies were used appropriately: BE0091, BE0087, BP0090, MOPC-21, or HPRN. All neutralizing and control antibodies were from BioXCell. For intracranial co-treatment of SMC and type I IFN, mice were injected i.t. with 10 μL of vehicle (0.5% DMSO), 100 μM LCL161, 0.01% BSA, or 1 μg IFN-α B / D combination. Alternatively, mice were treated orally with vehicle or 75 mg / kg LCL 161 and with 1 μg IFN-α B / D (ip). The animals were euthanized when they showed predetermined signs of neuropathy (non-walkable, weight loss greater than 20%, inactivity, feline back posture). Treatment groups were assigned per cage, each group having 5-9 mice for statistical scale (Kaplin-Meier with log rank analysis). There was no randomization, and the investigator was blinded to group assignment.

  Although statistical methods were not used to determine sample size, sample size is consistent with previous reports of examining tumor growth and mouse survival after cancer treatment.

MRI
Live mouse brain MRI was performed at the University of Ottawa preclinical imaging core using 7 Tesla GE / Agilent MR 901. The mice were anesthetized for MRI procedures using isoflurane. 2D fast spin echo sequence (FSE) pulse sequence, the following parameters: 15 predetermined slices, slice thickness = 0.7 mm, spacing = 0 mm, field of view = 2 cm, matrix = 256 × 256, echo time = 25 ms, repetition Time = 3,000 ms, echo training length = 8, bandwidth = 16 kHz, 1 average, and fat saturation were used for imaging. FSE sequences were performed in both the cross-section and the frontal plane for a total imaging time of about 5 minutes.

EMT 6 mammary tumor model
Mammary tumors were established by injecting 1 × 10 ⁵ EMT6 cells into the mammary fat pad of 5-week-old female BALB / c mice. Mice with palpable tumors (approx. 100 mm ³ ) orally with vehicle (30% 0.1 M HCl, 70% 0.1 M NaOAc pH 4.63) or 50 mg / kg LCL 161 and 5 × 10 ⁸ PFU Were co-treated with it injection of VSVΔ51 or ip injection of control IgG (BE0091) or anti-PD-1 antibody (J43). If the tumor has spread into the abdominal cavity, or tumor burden if it exceeds 2,000 mm ^3, animals were euthanized. Tumor volume was calculated using (π) (W) ² (L) / 4, where W = the width of the tumor and L = the length of the tumor. Treatment groups were assigned per cage and each group had 4-5 mice on a statistical scale (mean, standard error; Kaplan-Meier using log rank analysis). There was no randomization, and the investigator was blinded to group assignment.

MPC-11 Multiple Myeloma Model
A mouse model of multiple myeloma and plasmacytoma was established by injecting 1 × 10 ⁶ luciferase tagged MPC-11 cells (iv) into female 4-5 week old BALB / c mice. Mice are treated orally with vehicle (30% 0.1 M HCl, 70% 0.1 M NaOAc pH 4.63) or 75 mg / kg LCL 161 and with 250 μg control IgG or α-PD-1 antibody (ip) did. After ip injection of 4 mg luciferin (Gold Biotechnology), bioluminescence imaging was captured using a Xenogen 2000 IVIS CCD-camera system (Caliper Life Sciences). Treatment groups were assigned per cage, and each group had 3 to 4 mice on a statistical scale (Kaplin-Meier with log rank analysis). There was no randomization, and the investigator was blinded to group assignment.

Tumor re-challenge: naive age-matched female C57BL / 6 mice or mice that had previously been cured of intracranial CT-2A tumor by combined treatment based on SMC with immunostimulant (minimum 180 days after implantation) CT-2A cells as described above were reinjected ic or 5 × 10 ⁵ cells subcutaneously. 5 × 10 ⁵ untagged EMT 6 cells in the fat pad of mice (90-120 days after implantation) where luciferase-tagged EMT 6 mammary tumors have previously been cured by treatment with naive BALB / c or SMC and VSV Δ51. I was injected again. The animals were euthanized as described above. Blinding or randomization was not possible. All animal experiments were conducted with the approval of the University of Ottawa Animal Care and Veterinary Service, according to the guidelines established by the Canadian Council on Animal Care.

Flow cytometry
For in vitro analysis, cells were treated with vehicle (0.01% DMSO) or 5 μM LCL 161 and 0.01% BSA, 1 ng / mL TNF-α, 250 U / mL IFN-β or 0.1 MOI VSV Δ51 for 24 hours It was processed. Cells were released from the plate using enzyme-free dissociation buffer (Gibco) and stained with Zombie Green and the indicated antibodies. For analysis of tumor immune infiltrates, intracranial CT-2A tumors were mechanically dissociated, RBCs were dissolved in ACK lysis buffer and stained with Zombie Green and the indicated antibodies. In some cases, cells were stimulated with 5 ng / ml PMA and 500 ng / ml ionomycin for 5 hours in the presence of brefeldin A and intracellular antigens were processed using the BD Cytofix / Cytoperm kit. The antibodies may be Fc block (101319, 1: 500), PD-L1 (10F.9G2, 1: 250), PD-L2 (TY25, 1: 100), IA / IE (M5 / 114.15.2, 1: 200). And H-2 Kd / H-2 Dd- (34-1-2S, 1: 200), CD45 (30-F11, 1: 300), CD3 (17A2, 1: 500), CD4 (GK 1.5, 1: 500), CD8 (53-6.7, 1: 500), PD-1 (29.1 A12, 1: 200), CD25 (PC 61, 1: 150), Gr1 (RB6-AC5, 1: 200), F4 / 80 BM8, 1: 200), GrzB (GB 11, 1: 150) and IFN-γ (XMG 1.2, 1: 200). All antibodies were from BioLegend, except TNF-α (MP6-XT22, 1: 200) and CD11b (M1 / 70, 1: 100) from BD Biosciences. Cells were analyzed on Cyan ADP9 (Beckman Coulter) or BD Fortessa (BD Biosciences) and data were analyzed using FlowJo (Tree Star).

Microscopic observation
Detection of mKate2-CT-2A cells was performed in an incubator equipped with an Incucyte Zoom microscope equipped with a 10 × objective. The counts of fluorescence signal from Inuccyte Zoom were processed using the integrated object counting algorithm in Inuccyte Zoom software.

Multiplex ELISA
Detection of serum proteins after combined treatment of SMC and anti-PD-1 antibody was analyzed by flow cytometry based multiplex kit (LEGENDplex inflammation panel from Biolegend). Hierarchical analysis was determined using Morpheus (https://software.broadinstitute.org/morpheus).

RT-qPCR
Total RNA was extracted from cells using the RNeasy mini prep kit (Qiagen). Two-step RT-qPCR was performed on a Mastercycler ep realplex (Eppendorf) using iScript and SsoAdvanced SYBR Green supermix (BioRad). qPCR was performed using PD-L1 and PD-L2 primers (Qiagen) and SIBR green reagent (Bio-Rad). The relative expression was calculated as ΔΔCt using RPL13A as a control.

  The library panel of cytokine and chemokine genes was from realtimeprimers.com. For each conditioned treatment, n = 4 was performed and data were normalized to eight different reference genes and compared to the respective vehicle and IgG samples. Data were analyzed by hierarchical analysis using Morpheus.

ELISpot
Female age-matched na ナ イ ve mice or mice with previously cured intracranial CT-2A (180 days post implantation) or mammary gland EMT 6 tumors (120 days post implantation) using the CD8 magnetic selection kit (Stemcell Technologies) Splenocytes were enriched for CD8 + T cells. CD8 + cells were transformed with cancer cells (1:20 for CT-2A, LLC and 1: 12.5 for EMT6 or 4T1 cells) using IFN-γ or Granzyme B ELISpot kit (R & D Systems). And co-cultured with 10 μg / mL of IgG (BE0091) or anti-PD-1 antibody (J43) for 48 hours.

Statistics For all animal studies, survival was calculated from the number of days post MM cell implantation and plotted as a Kaplan-Meier curve. From these, the log rank test was used to determine significance. Errors are presented as standard deviations for in vitro viability assays. Subsequent pairwise multiple comparisons were performed using the Form-Sidak method (SigmaPlot). Comparisons between multiple treatment groups were analyzed using one-way ANOVA followed by post hoc analysis using Dunnett's multiple comparison test with adjustment for multiple comparisons (GraphPad). Estimates of variability were analyzed using GraphPad. Treatment pair comparisons were analyzed by two-tailed t-test (GraphPad).

(Example 5)
Combination of Immunostimulatory Agents for Glioblastoma Therapy We have found that cultured and primary glioblastoma cell lines are exogenous TNF-α, oncolytic virus VSVΔ51, or infectious but not replicative. When combined with the virus VSVΔ51ΔG, it is shown that it is killed by SMC (FIGS. 46A and 46B). We observed cell death of glioblastoma cells using a combination of TNF-α and various SMCs, so the synergistic effect between SMC, LCL161 and TNF-α is within this drug class It confirmed that it was a general phenomenon (FIG. 47). Furthermore, we also observed enhancement of SMC efficacy using oncolytic Rhabdovirus, VSVΔ51 or Maraba-MG1 on human brain tumor initiating cells (BTIC) (FIG. 46C). Non-replicating Rhabdovirus particles (NRRP), which retain their infectivity and immunostimulatory properties without the ability to replicate, were also found to synergize with SMC to induce glioblastoma cell death . Notably, only about 50% of the profiled cancer cell lines are sensitized to cell death in combination with SMC and TNF-α or TNF related apoptosis inducing ligand (TRAIL); most Resistant cell lines are further sensitized to cell death with downregulation of the caspase 8 inhibitor, cFLIP (cellular FLICE-like inhibitor protein). Consistent with these previous findings, two glioblastoma cell lines that were refractory to combined treatment with SMC and TNF-α or VSVΔ51ΔG were killed upon cFLIP silencing (Figure 48A and 48B). In contrast, normal diploid human fibroblasts were not sensitized to cell death with downregulation and combination treatment of cFLIP. These findings suggest that IFN and / or cytokine responses, rather than direct virus-induced cell lysis, are responsible for SMC-induced cell death of glioblastoma cells.

  Because VSV Δ51 is neurotoxic and there is still a problem with the brain's microenvironment with “immune privileges” and the penetration of drugs across the blood-brain barrier (BBB), we therefore have systemic and intracranial immunity. We tried to test the effect of therapeutic agent delivery. After establishment of the intracranial CT-2A tumor (FIGS. 49A and 49B), we demonstrate that systemic administration by oral gavage of SMC, LCL 161 is its major target protein cIAP1, within the intracranial mouse tumor. And whether it could cause transient degradation of cIAP2. In contrast, we did not observe downregulation of cIAP, either in the nearby non-neoplastic brain tissue or in the cortex or cerebellum in non-tumor bearing mice (FIG. 51). Thus, SMCs have the ability to reach tumors in the brain where the BBB has been damaged. Systemic administration of an immunostimulant such as poly (I: C), a synthetic TLR3 agonist injected intraperitoneally (ip) or the oncolytic virus VSV Δ51 administered intravenously (iv), results in non-tumor bearing mice Induced the production of the cytokine TNF-α in the serum and brain of

  Prolonged survival of mice when mice bearing intracranial CT-2A glioblastoma are treated alone with SMC (oral gavage), VSV Δ51 (iv) m or poly (I: C) (intracranial, ic) Was minimal for this aggressive cancer (17% survival rate) (FIG. 51C). However, the combination of systemic SMCs with the immunostimulatory agent, VSVΔ51 or poly (I: C) significantly prolonged survival, resulting in permanent cure for 71% or 86% mice, respectively. Tumors (not tagged with foreign proteins to avoid immune enhancement) were imaged by MRI at day 40 after implantation to confirm the outcome of the observed treatment.

  Virus-induced immune effects are, in part, mediated by type I IFN. We show that CT-2A cells are partially sensitive to the combination of SMC and recombinant IFN-α in vitro (FIG. 50A). We observed that intracranial administration of SMC resulted in even more pronounced degradation of IAP protein in CT-2A brain tumors (FIG. 53). For in vivo testing, we have shown recombinant IFN-α consisting of a hybrid of human isoforms IFN-αB and IFN-αD that exhibits potent antiviral activity among a wide range of species It was used. A single co-administration of SMC and IFN-α significantly prolonged the survival of mice, resulting in a permanent cure rate of 50%. Long-term survival mice showed no apparent physical or behavioral deficits from single or combined intracranial treatment of SMC, poly (I: C) or IFN-α (FIG. 54). Furthermore, because we observed a transient increase in intracranial TNF-α in the brain upon treatment with systemic VSV Δ51 infection or poly (I: C), we treated with SMC treatment We sought to determine if simultaneous systemic administration of recombinant IFN-α was effective in the CT-2A glioblastoma model. Similar to the combination of SMC and VSVΔ51, the combination of IFN-α administered ip with oral gavage of SMC resulted in permanent cure in 55% of the mice (FIG. 50B). These results suggest that the presence of a transient inflammatory environment in the brain is tolerated, and indirect and other direct (intracranial) routes of administration of combination treatments may be feasible It is shown that.

(Example 6)
Generation of Long-Term Tumor Immunity in Cured Mice The innate immune system is central to SMC-mediated cell death of tumor cells. Nevertheless, there remain fundamental questions about the contributing role of the adaptive immune system in this SMC combinatorial approach. Furthermore, a potential pitfall of the proposed use of oncolytic viruses or other immunostimulants in combination with SMC treatment is the increased expression of checkpoint inhibitor ligands on cancer cells, thereby causing CTL in the tumor. It may be the cancellation of a vectorial attack. By flow cytometric analysis, treatment of glioma cells with recombinant type I IFN or infection with VSV Δ51 but not treatment with TNF-α resulted in surface expression of PD-L1 and major histocompatibility complex (MHC) I markers It has been shown to bring about an increase. Furthermore, SMC treatment had no significant effect on the expression of these tumor surface molecules (FIG. 52A and FIG. 56).

  Interestingly, mice that had previously cured orthotopic EMT6 mammary adenocarcinoma by SMC combination treatment were completely resistant to tumor engraftment when EMT6 cells were re-challenged (Figure 52B). However, another syngeneic cell line 4T1, which shares major histocompatibility proteins, was not rejected from these cured mice. We found that mice in which the intracranial CT-2A tumor was cured were also resistant to tumor engraftment of CT-2A cells injected subcutaneously or intracranially (FIG. 52C). We next evaluated the cytotoxic potential of CD8 T cells from cured mice by ELISpot assay. The stimulation by CT-2A cells of CD8 + T cells from cured mice but not cells isolated from naive mice is specific as indicated by enhanced IFN-γ and granzyme B (GrzB) production Showed the presence of reactive T cells (FIG. 54A). The inclusion of anti-PD-1 blocking antibody further increased the expression of IFN-γ and GrzB. Similar results were observed for mice that had EMT6 tumors cured (FIG. 44D). Taken together, these results suggest the generation of robust and specific long-term tumor immunity using SMC combination therapy.

(Example 7)
Immune Checkpoint Inhibitors Synergize with IAP Antagonists Next, Can the Current Class of Cancer Immunotherapy, Known as Immune Checkpoint Inhibitors or ICI, Enhance SMC Efficacy? I investigated. It has recently been reported that ICI treatment of glioblastoma in mice results in at least partial prolongation of survival. We first sought to determine whether SMC treatment affects PD-1 expression in a subset of infiltrating immune cells within CT-2A brain tumors. Although there were no statistical differences between the levels of infiltrating CD3 + or CD3 + CD8 + cells within the intracranial CT-2A tumor, we did not detect immune checkpoints, CD-1 and CD3 + CD8 + cells expressing PD-1. A robust increase was observed (FIGS. 54B and 55). There was an overall increase in expression of PD-L1 in CD25- cells, mainly CT-2A cells, but the trend did not reach statistical significance (FIG. 54C).

  To determine if increased levels of PD-1 + CD8 T cells are a negative regulator of SMC efficacy, we used two mouse models of glioblastoma Blockade of the checkpoint target, PD-1 and CTLA-4 in combination with was assessed. Systemic administration of anti-PD-1 antibody or anti-CTLA4 antibody showed no activity against itself (FIG. 54D and FIG. 54E). In contrast, the combination of anti-PD-1 antibody and SMC significantly prolonged survival in the CT-2A and GL261 models, respectively, resulting in permanent cure rates of 71% and 33%. Furthermore, when combined with SMC, anti-PD-1 biologic was superior to anti-CTLA-4 biologic in the CT-2A model (71% vs. 43%; FIG. 54D).

  There are two structural classes of SMC: monomers and dimers. Monomeric SMCs consist of a single chemical molecule that binds to the BIR domain of IAP, but dimeric SMCs are two SMCs connected by a linker that allows for cooperative binding and / or anchoring of IAPs. It consists of molecules. LCL 161, a clinically advanced SMC, is the focus of many of our studies and is a potent monomer. We next sought to evaluate whether another clinically advanced dimeric SMC would be as synergistic as ICI for the treatment of glioblastoma. We observed a significant increase in the survival of mice bearing intracranial CT-2A tumors when treated with anti-PD-1 antibody and the dimeric SMC, Viliampant (FIG. 54F). Since combined blockade of PD-1 or CTLA-4 is beneficial for patients with melanoma, do we combine the combination of PD-1 with CTLA-4 to significantly enhance SMC therapy as well? I tried to decide. The combination of antibodies targeting PD-1 and CTLA-4 is effective in inducing permanent cure in a mouse model of cancer and we observed an overall survival of 67% (FIG. 54G). Surprisingly, with SMC treatment and the inclusion of anti-PD-1 and anti-CTLA-4 antibodies, a 100% permanent cure rate was obtained.

  The synergistic effect of SMC and ICI is not limited to brain tumors. We also used MPC-11 cells to observe a significant prolongation of the survival of mice bearing a highly aggressive and treatment refractory model of multiple myeloma (FIGS. 56A and 56B). A permanent cure rate of 75% was also observed in mice bearing mammary gland EMT6 tumors, which was further increased to 100% with the inclusion of immunostimulatory agents (Figures 57A-C).

(Example 8)
CD8 + T cells are required for the efficacy of SMCs and ICI To provide the first insight in the cellular mechanism of action we used a combination treatment of SMCs and anti-PD-1 antibodies. The production of immune factors from CT-2A cells cocultured with splenocytes from intracranial CT-2A tumor cured mice was profiled. We observed a significant increase in IFN-γ and GrzB production from CT-2A cells co-cultured with splenocytes from surviving mice (FIG. 58A and FIG. 59A). Of note, there was an increase in the production of interleukin 17 (IL-17). We also observe reduced expression of the proinflammatory cytokines IL-6 and TNF-α, considering that IL-17 has been previously found to stimulate the NF-κB pathway However, this was unexpected.

  However, the expression of IFN-γ and IL-17 from splenocytes isolated from cured mice was significantly increased by anti-PD-1 or PD-L1 treatment, but this is due to the T cell-based immune response Suggest that it can be increased upon checkpoint inhibition by the PD-1 axis. We next sought to determine if this gene response was affected by SMC treatment. Among previously analyzed cytokines, inclusion of SMC in these co-cultures with anti-PD-1 blockers increased secretion of IFN-γ, GrzB, IL-17, and TNF-α ( Figures 59B and 59B). Notably, the levels of IL-6 in the supernatant were not affected by SMC treatment. Furthermore, the immunosuppressive cytokine IL-10 had a general tendency to decrease secretion upon combined treatment of SMC and anti-PD-1.

  We now have glioblastoma cells and naive mice or CT-2A intracranial tumors due to increased levels of GrzB, a cytotoxic factor partially blocked by XIAP 31-33 and TNFα. Whether co-culture with splenocytes from previously cured mice resulted in cell death of CT-2A cells was assessed. Using SMCs of different structures, we saw a statistically significant increase in cell death of CT-2A cells in the presence of SMCs, but this response was of anti-PD-1 antibody It increased with the content (FIG. 59C).

  Taken together, these results indicate that a robust effector T cell response is elicited using the combined treatment of ICI and SMC. To further elucidate the cellular mechanism of action, we performed immune cell depletion using specific CD4 or CD8 targeting antibodies. We found that the 71% cure rate induced by combination therapy was completely abolished upon depletion of CD8 + T cells (FIG. 59D). Interestingly, depletion of CD4 + T cells resulted in a 100% cure rate for the combination of SMC and anti-PD-1 antibody, and a 17% cure rate in the control group. These results suggest that removal of CD4 + immunosuppressive cells (such as regulatory T cells) helps to induce tumor regression and that CD4 + cells are not required for the efficacy of the combination treatment approach. In a second approach, intracranial CT-2A tumors were established in CD1 nude mice lacking functional T cells and then treated with anti-PD-1 antibodies in combination with vehicle or SMC. The survival benefit provided by the combination of SMC and anti-PD-1 antibody was lost in these T cell deficient mice (FIG. 59E). Overall, the synergy between SMC and anti-PD-1 antibodies relies on a functional adaptive immune response, thus involving CD8 + T cells as primary immune cell mediators for in vivo efficacy.

(Example 9)
SMC treatment affects intratumoral immune cell infiltration
To understand the immunocytological aspects of the synergy between SMC and ICI treatment, we evaluated the profile of infiltrating CD45 + immune cells in mice bearing glioblastoma. In these studies, we evaluated infiltrating immune cells in late stage glioblastoma tumors after co-treatment with anti-PD-1 antibody and SMC (FIG. 61A). Flow cytometric analysis of tumor infiltrating T cells tends to be not statistically significant in the proportion of CD4 + and CD8 + T cells between vehicle and IgG control treated groups and all single and double treated mice Shown (FIG. 61B). However, analysis of CD4 + and CD25 + T cells, showing a regulatory T cell (Treg) population, showed a significant reduction of this cell population with respect to SMC treatment alone or a combination of SMC and ICI (FIG. 61C) ).

  Next, we characterized the surface presentation of PD-1 on T cells after single and combined treatment. We found a significant increase in CD8 + T cells expressing PD-1 in mice treated with SMC only, anti-PD-1 antibody treatment, or a combination of SMC and anti-PD-1 antibody treatment. It was noted that it resulted in a less detectable surface presentation of PD-1 (FIG. 61D). Furthermore, we observed a trend towards reduced presentation of PD-1 in CD4 + T cells in the SMC or anti-PD-1 treated groups. However, detectable levels of surface PD-1 were abolished using a combination treatment of SMC and anti-PD-1 (FIG. 61E).

  In addition to the observed T cell infiltration of intracranial glioblastoma tumors, we next characterized the presence of bone marrow derived suppressor cells (MDSC) and astrocytes / microglia. In contrast to previous reports, we did not detect differences in the MDSC population (CD11b + Gr1 +) in all treatment cohorts (FIG. 61F). However, we noticed that the astrocyte / microglia population is significantly reduced in the treatment cohort containing anti-PD-1 antibody (FIG. 61G). Overall, these results indicate that the combined treatment result is a reduction in the immunosuppressive CD4 T cell population, with a concomitant decrease in PD-1 presentation in T cells and a reduction in astrocytes and / or microglia. Indicates that.

(Example 10)
Synergism between SMC and ICI is dependent on TNF-α We next found that the tumor cell nature of mice bearing intracranial glioblastoma tumors treated with a combination of SMC and anti-PD-1 antibody Cytokine and chemokine profiles were characterized. Flow cytometric analysis showed that there was an increase in CD8 + cells expressing GrzB with the inclusion of anti-PD-1 antibody. The ratio of cytotoxic CD8 + (FIG. 62A) to CD4 + Treg was also increased in the anti-PD-1 antibody and SMC and anti-PD-1 antibody treated cohort (FIG. 62B). In addition to assessing GrzB expression, we analyzed the levels of IFN-γ and TNF-α in T cells. Unexpectedly, we observed a decrease in the proportion of CD4 + cells expressing IFN-γ during SMC treatment (even in the presence of antibodies targeting PD-1), but within CD8 + cells No change in IFN-γ expression levels was seen in any of the treatment cohorts (FIG. 62C). We then analyzed the expression level of TNF-α in T cells. In this context, we observed a significant increase in TNF-α expressing CD4 + and CD8 + T cells (Figure 62D), which indicates that these T cells do not mediate SMC-mediated tumor cell death. It shows that it can be directly derived.

  We also evaluated the effect of the combination of SMC treatment and an anti-PD-1 antibody blocker on serum concentrations and gene expression levels of cytokines and chemokines in an intracranial CT-2A glioblastoma model. We show statistically significant increases in the proinflammatory cytokines IFN-, IL-1-alpha, IL-1 beta, and IL-17 and the pleiotropic cytokines IFN-gamma, IL-27 and GM-CSF. Were detected (FIG. 60 and FIG. 62E). Of note, there was no difference in the presence of anti-inflammatory cytokines such as IL-10. Similarly, analysis of cytokine and chemokine expression profiles within intracranial CT-2A tumors after combined treatment with SMC and ICI showed clustering of proinflammatory cytokines and chemokines (FIG. 62F and FIG. 63). These candidates derived from SMC or a combination treatment of SMC and ICI are proinflammatory cytokines IFN-β, IL-1β, IL-17, Osm, and TNF-α, the chemokine Ccl2 (also known as MCP-1) ), Ccl5, Ccl7, Ccl22, Cxcl9, Cscl10 and Cxcl11, and pleiotropic factors such as FasL, IL-2, IL-12 and IFN-γ.

  Since we observed a consistent increase in the levels of IFN-β and IFN-γ, we next carried intracranial CT-2A tumors and were treated with SMC and anti-PD-1 In mouse mice, blocking / neutralizing antibodies were used to characterize the functional role of these signaling molecules. Abolition of type I IFN signaling by using antibodies that block the IFNAR1 receptor has a synergistic effect towards increasing the survival of mice bearing intracranial CT-2A tumors after combined treatment with SMC and anti-PD1 antibody Is disabled (FIG. 62G). In contrast, antagonism of IFN-γ function by using an anti-IFN-γ antibody partially inhibited the synergistic effects of combination treatment of SMC and ICI. Overall, these results indicate that each treatment, even when combined, results in the generation of different gene and protein signatures, but generally rely on intact type I IFN signaling.

  Overall, our results indicate that the synergistic effect of SMC and ICI can be mainly due to the enhancement of CTL-mediated attack on glioblastoma cells, which is proinflammatory including type I IFN Include a response. Co-culture of CT-2A cells with CD8 + T cells isolated from mice where intracranial tumors had previously cured resulted in an increase in GrzB positive CD8 + T cells, but this was only due to SMC treatment It did not increase (FIG. 65A). However, even when the PD-1 / PD-L1 axis was abolished, viable CT-2A cells were only slightly reduced when cocultured with the same CTL (FIG. 65B). Since we were previously aware that the type I IFN response also results in the production of TNF-α, we produce T-TN after α.T treatment in the presence of glioblastoma cells. The capacity of the cells was assessed. Therefore, we next assessed the production of TNF-α.

  The inclusion of SMC significantly increased the proportion of CD8 + T cells expressing TNF-α regardless of the inclusion of antibodies targeting PD-1 (FIG. 65A). As the expression level of TNF-α from CD8 T cells increased, we significantly reduced CT-2A cells in coculture system using CT-2A cells and CD8 T cells derived from cured mice Was observed (FIG. 65B). Of note, SMC-mediated effects on triggering cell death of CT-2A cells were mainly dependent on TNF-α, the main mediator of SMC-induced tumor killing. Next, we assessed whether SMC treatment enhanced T cell proliferation. Indeed, after co-incubation of CT-2A cells, we observed a significant decrease of CFSE loaded CD8 + T cells with the appearance of a new population of faintly labeled CFSE cells, this effect Notable for the inclusion of SMC and anti-PD-1 antibody (FIG. 64).

  These results demonstrate that cytotoxic T cells, a cell death promoter that induces tumor cell death due to antagonism of IAP in response to treatment with SMC and anti-PD-1 antibody, GrzB and TNF FIG. 16 shows that increased tumor cell death may result due to increased production of -α. We functionally characterized the role of TNF-α by using blocking antibodies that target TNF-α. When systemic blockade of TNF-α was added, we observed almost complete reversal of the efficacy of combination treatment with SMC and ICI (Figure 65C), which is a synergy of these different agents It highlights the importance of TNF-α for its effect.

  The immunomodulatory anti-cancer effects of SMC are diverse (Figure 66 and Figure 67). SMC can polarize macrophages from the immunosuppressive M2 type towards the M1 phenotype producing inflammatory TNF-α. Furthermore, SMC anti-cancer effects are highly promoted by proinflammatory cytokines, and the presence of these cytokines, such as TNF-α or TRAIL, in the tumor microenvironment results in tumor cell death. Specifically, SMC-mediated depletion of cIAP changes TNF-α-mediated survival responses into cell death pathways in cancer cells.

  Our current studies show that SMC can dramatically enhance and cooperate with the action of ICI, including anti-PD-1 or anti-CTLA4 antibodies, mice bearing aggressive intracranial tumors. Prove that it allows for a permanent cure of The multiplicity and complexity of mechanisms associated with SMC therapy make it difficult to isolate the individual roles involved in changing immunoregulatory activity in combination synergy. However, it is clear that the cytotoxicity of TNF-α is involved. Furthermore, current studies further demonstrate that CD8 + T cells are also required for anti-cancer activity when ICI is combined with SMC.

  Taken together, we have shown for the first time that SMC can promote the activity of ICI in mouse tumor models. Furthermore, this combined effect is dependent on the presence of CD8 + T cells, with concomitant reductions in immunosuppressive CD4 + T cells and type I and II IFN and TNF-α signaling pathways, and SMC-mediated in mice Clearly show the role of adaptive immunity for sexual healing. Thus, SMC-mediated T cell costimulatory signals provide the drive for the adaptive immune response generated against the tumor, which is due to ICI that the brakes imposed by the coinhibitory signals, such as PD-1 or PD-L1 Completely realized if removed.

Other Embodiments All publications, patent applications, and patents described herein are incorporated herein by reference.

  While the invention has been described in conjunction with specific embodiments, it will be understood that further modifications are possible. Thus, the present application is generally any modification, use, or adaptation of the present invention in accordance with the principles of the present invention, including any departure from the present disclosure which is within known or routine practice in the art. Is intended to encompass.

Claims (46)

  (i) SMC derived from Table 1 (Table 1) and (ii) Drug derived from Table 2 (Table 2) or Table 3 (Table 3) or drug that is an immune checkpoint inhibitor (ICI) or STING agonist And said SMC and said agent are provided together in an amount sufficient to treat cancer when administered to a patient in need thereof. .   A method for treating a patient diagnosed with cancer, comprising: (i) SMC derived from Table 1 (Table 1); (ii) Table 2 (Table 2) or Table 3 (Table 3) 28.) administering an agent derived from or an agent that is an ICI or a STING agonist, wherein the SMC and the agent together, in an amount sufficient to treat the cancer, or The method administered within a day.   3. The method of claim 2, wherein the SMC and the agent are administered within 14 days of each other.   5. The method of claim 3, wherein the SMC and the agent are administered within 10 days of each other.   5. The method of claim 4, wherein the SMC and the agent are administered within 5 days of each other.   6. The method of claim 5, wherein the SMC and the agent are administered within 24 hours of each other.   7. The method of claim 6, wherein the SMC and the agent are administered within 6 hours of each other.   8. The method of claim 7, wherein the SMC and the agent are administered substantially simultaneously.   9. The method according to any one of claims 2 to 8, wherein the SMC is a monovalent SMC.   10. The method of claim 9, wherein the SMC is LCL 161.   10. The method of claim 9, wherein the SMC is GDC-0152 / RG7419, or GDC-0917 / CUDC-427.   10. The method of claim 9, wherein the SMC is SM-406 / AT-406 / Debio 1143.   The method according to any one of claims 2 to 8, wherein the SMC is bivalent SMC.   14. The method of claim 13, wherein the SMC is AEG40826 / HGS1049.   14. The method of claim 13, wherein the SMC is OICR 720.   14. The method of claim 13, wherein the SMC is TL32711.   14. The method of claim 13, wherein the SMC is SM-1387 / APG-1387.   The method according to any one of claims 2 to 17, wherein the agent is a TLR agonist derived from Table 2.   19. The method of claim 18, wherein the agent is lipopolysaccharide, peptidoglycan, or lipopeptide.   19. The method of claim 18, wherein the agent is a CpG oligodeoxynucleotide.   21. The method of claim 20, wherein the agent is CpG-ODN 2216.   19. The method of claim 18, wherein the agent is imiquimod.   19. The method of claim 18, wherein the agent is poly (I: C).   19. The method of claim 18, wherein the agent is BCG.   The method according to any one of claims 2 to 17, wherein the agent is a virus derived from Table 3.   26. The method of claim 25, wherein the agent is Vesicular stomatitis virus (VSV).   27. The method of claim 26, wherein the agent is VSV-M51R, VSV-MΔ51, VSV-IFNβ, or VSV-IFNβ-NIS.   26. The method of claim 25, wherein the agent is adenovirus, malababe cyclovirus, reovirus, rhabdovirus, vaccinia virus, or a variant thereof.   26. The method of claim 25, wherein the agent is talimogene la harpa lepbek.   18. The method of any one of claims 2-17, wherein the agent is ICI.   The ICI is ipilimumab, tremelimumab, penbrolizumab, nivolumab, pizirizumab, AMP-224, AMP-514, AUNP12, PDR001, BGB-A317, REGN 2810, averumab, BMS-935559, atezolizumab, durvalumab, BMS-986016, LAG525, MBG453, MBG453 31. The method of claim 30, wherein the method is selected from the list consisting of: lirilumab, or MGA 271.   32. The method of any one of claims 2-31, wherein the cancer is refractory to treatment with SMC in the absence of drug.   33. The method of any one of claims 2-32, wherein the treatment further comprises the administration of a therapeutic agent comprising an interferon.   34. The method of claim 33, wherein the interferon is type 1 interferon.   The cancer includes adrenal cancer, basal cell cancer, biliary cancer, bladder cancer, osteosarcoma, brain cancer, breast cancer, cervical cancer, choriocarcinoma, colon cancer, colorectal cancer, connective tissue cancer, Gastrointestinal cancer, endometrial cancer, nasopharyngeal cancer, esophagus cancer, eye cancer, gallbladder cancer, stomach cancer, head and neck cancer, hepatocellular carcinoma, intraepithelial neoplasia, kidney cancer, Laryngeal cancer, leukemia, liver cancer, liver metastasis, lung cancer, lymphoma, melanoma, myeloma, multiple myeloma, neuroblastoma, mesothelioma, glioma, myelodysplastic syndrome, multiple myeloma, Oral cancer, ovarian cancer, childhood cancer, pancreatic cancer, pancreatic endocrine tumor, penile cancer, plasma cell tumor, pituitary adenoma, thymoma, prostate cancer, renal cell cancer, respiratory cancer, Selected from Rhabdomyosarcoma, salivary gland cancer, sarcoma, skin cancer, small intestine cancer, stomach cancer, testicular cancer, thyroid cancer, ureteral cancer, and urological cancer That A method according to any one of claims 2 34.   A composition comprising an SMC derived from Table 1 and an agent, wherein the agent comprises a killed virus, an inactivated virus, or a viral vaccine, wherein the SMC and the agent require it. A composition which, when administered to a patient, is provided in an amount sufficient to treat cancer together.   37. The composition of claim 36, wherein the agent is NRRP or a rabies vaccine.   A composition comprising an SMC derived from Table 1 and an agent, wherein the agent comprises a first agent that primes an immune response, and at least a second agent that boosts the immune response, A composition wherein the SMC and the agent are provided together in a sufficient amount to treat cancer when administered to a patient in need thereof.   39. The composition of claim 38, wherein one or both of the first agent and the second agent are oncolytic virus vaccines.   40. The composition of claim 39, wherein the first agent is an adenovirus carrying a tumor antigen and the second agent is a becyclovirus.   39. The composition of claim 38, wherein the veciclovirus is selected from Maraba-MG1 carrying the same tumor antigen as the adenovirus and Maraba-MG1 not carrying the tumor antigen.   A composition comprising SMC and ICI, wherein said SMC and said ICI are provided in an amount sufficient to treat cancer together when administered to a patient in need thereof. Composition.   43. The composition of claim 42, wherein the SMC is an SMC derived from Table 1 (Table 1).   44. The composition of claim 42 or 43, wherein the ICI is an ICI derived from Table 4.   (i) a composition comprising SMC derived from Table 1 and (ii) two, three, four, three or more agents, wherein each agent is independently ICI, or A composition which is an agent derived from Table 2 or an agent derived from Table 3 or is a STING agonist.   46. The composition of claim 45, wherein the ICI is an ICI derived from Table 4.